Compositions containing hc-ha/ptx3 complexes and methods of use thereof

ABSTRACT

Provided herein are methods for the production of native and reconstituted hyaluronan (HA) complexes containing pentraxin-3 (PTX3) and heavy chain 1 (HC1) of inter alpha inhibitor (IαI). Compositions containing the complexes and therapeutic methods using the complexes are provided. Combinations and kits for use in practicing the methods also are provided.

CROSS-REFERENCE

This application is a continuation of U.S. Application No. 16/898,309,filed Jun. 10, 2020, which is a continuation of U.S. Application No.16/274,123, filed Feb. 12, 2019, which is a continuation of U.S.Application No. 15/214,706, filed on Jul. 20, 2016, now issued as U.S.Pat. No. 10,253,065 on Apr. 9, 2019, which is a continuation of U.S.Application No. 14/414,047, filed Jan. 9, 2015, now issued as U.S. Pat.No. 10,040,821 on Aug. 7, 2018, which is the National Phase Entry ofInternational Application No. PCT/US2013/049983, filed Jul. 10, 2013,which claims the benefit of U.S. Provisional Application No. 61/670,571,filed on Jul. 11, 2012, all of which are hereby incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under RO1 EY06819, R44EY017497, and R43 EY021045 awarded by the National Eye Institute, theNational Institutes of Health, Bethesda, MD, USA. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in XML format and is hereby incorporated by reference in itsentirety. Said XML copy, created on Nov. 1, 2022, is named“34157-832-304_SL.txt” and is 139,395 bytes in size.

BACKGROUND OF THE INVENTION

The amniotic membrane (AM) is an avascular membranous sac that is filledwith amniotic fluid surrounding the fetus. The AM, like the placenta, isderived from the epiblast formed during development of the fertilizedegg. The AM forms the innermost membrane surrounding the fetus in theamniotic cavity. In placental mammals, the umbilical cord (i.e., thefuniculus umbilicalis) connects the developing fetus to the placenta.The umbilical cord is made up of amniotic membrane (UCAM) and Wharton’sJelly. The amniotic membrane forms the outer layer of the umbilicalcord. The UCAM functions to regulate the fluid pressure within the UC.Wharton’s Jelly is a gelatinous substance within the umbilical cord,largely made up of mucopolysaccharides (hyaluronic acid and chondroitinsulfate). It also contains some fibroblasts and macrophages. Theumbilical cord further comprises two arteries (the umbilical arteries)and one vein (the umbilical vein), buried within the Wharton’s Jelly.

SUMMARY OF THE INVENTION

Described herein are methods for the identification of HC-HA/PTX3complexes in fetal tissues, such as amniotic membrane and umbilicalcord. Also, described herein are methods for the isolation of nativeHC-HA/PTX3 complexes from fetal tissues, such as amniotic membrane andumbilical cord. Also described herein are methods for the production ofreconstituted HC-HA/PTX3 complexes. Also described herein are methodsfor the use of native and reconstituted HC-HA/PTX3complexes providedherein.

Described herein, in certain embodiments, are methods of producingHC-HA/PTX3 complexes. In some embodiments, the methods compriseisolating a native HC-HA/PTX3 (nHC-HA/PTX3) complex from an extractprepared from a tissue or cell. In some embodiments, the methodscomprise generating a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex.

Described herein, in certain embodiments, are methods of isolatingnative HC-HA/PTX3 (nHC-HA/PTX3) complexes from amniotic tissues, such asumbilical cord or amniotic membrane. In some embodiments, thenHC-HA/PTX3 complexes are isolated from an isolated cell. In someembodiments, the nHC-HA/PTX3 complexes are isolated from a culturedcell. In some embodiments, the nHC-HA/PTX3 complexes are isolated from astem cell. In some embodiments, the nHC-HA/PTX3 complexes are isolatedfrom a water soluble fraction of an extract prepared from a tissue, suchas umbilical cord or amniotic membrane. In some embodiments, the watersoluble fraction is extracted with an isotonic salt solution. In someembodiments, the nHC-HA/PTX3 complexes are isolated from a waterinsoluble fraction of an extract prepared from a tissue, such asumbilical cord or amniotic membrane. In some embodiments, the insolublefraction is extracted with GnHCl.

Described herein, in certain embodiments, are methods of producing areconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, (a) contacting(i) high molecular weight hyaluronan (HMW HA) immobilized to a solidsupport, and (ii) pentraxin 3 (PTX3) protein, to form an immobilizedcomplex of PTX3 and HMW HA (immobilized PTX3/HA); and (b) contacting theimmobilized PTX3/HA with an inter-α-inhibitor (IαI) protein comprisingheavy chain 1 (HC1) and Tumor necrosis factor α-stimulated gene 6(TSG-6) to form an immobilized rcHC-HA/PTX3 complex. In someembodiments, steps (a) and (b) of the method are performed sequentiallyin order. In some embodiments of the method, the method comprisescontacting high molecular weight hyaluronan (HMW HA) with a pentraxin 3(PTX3) protein, inter-α-inhibitor (IαI) protein comprising heavy chain 1(HCl) and Tumor necrosis factor α-stimulated gene 6 (TSG-6)simultaneously. In some embodiments of the method, TSG-6 catalyzes thecovalent linkage of IαI HC1 to HA. In some embodiments, the methodfurther comprises removing unbound PTX3 protein following step (a) andprior to performing step (b). In some embodiments, the method furthercomprises removing unboundTSG-6 following step (b). In some embodiments,the PTX3 protein used in the methods is a native PTX3 protein isolatedfrom a cell. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a human cell. In some embodiments, the cell isan amniotic membrane cell. In some embodiments, the cell is an umbilicalcord cell. In some embodiments, the cell is an amniotic membrane cellfrom an umbilical cord. In some embodiments, the amniotic membrane cellis an amniotic epithelial cell. In some embodiments, the amnioticmembrane cell is an umbilical cord epithelial cell. In some embodiments,the amniotic membrane cell is an amniotic stromal cell. In someembodiments, the amniotic membrane cell is an umbilical cord stromalcell. In some embodiments, the PTX3 protein is a recombinant protein. Insome embodiments, the PTX3 protein used in the methods comprises apolypeptide having the sequence set forth in SEQ ID NO: 33 or apolypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence amino acid identity to the polypeptide having the sequenceset forth in SEQ ID NO: 33. In some embodiments, the PTX3 protein usedin the methods comprises a polypeptide having the sequence set forth inany of SEQ ID NOS: 32-45 or a species variant or allelic variantthereof. In some embodiments, the PTX3 protein used in the methods is amultimeric protein. In some embodiments, the PTX3 protein used in themethods is a homomultimer (i.e. a multimeric protein consisting of twoor more identical components). In some embodiments, the PTX3homomultimer is a dimer, trimer, tetramer, pentamer, hexamer, oroctamer. In some embodiments, the PTX3 homomultimer is an octamer. Insome embodiments, the PTX3 protein comprises a modified multimerizationdomain or a heterogeneous multimerization domain. In some embodiments,the TSG-6 protein used in the methods is a native TSG-6 protein isolatedfrom a cell. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a human cell. In some embodiments, the cell isan amniotic membrane cell. In some embodiments, the cell is an umbilicalcord cell. In some embodiments, the cell is an amniotic membrane cellfrom an umbilical cord. In some embodiments, the amniotic membrane cellis an amniotic epithelial cell. In some embodiments, the amnioticmembrane cell is an umbilical cord epithelial cell. In some embodiments,the amniotic membrane cell is an amniotic stromal cell. In someembodiments, the amniotic membrane cell is an umbilical cord stromalcell. In some embodiments, the TSG-6 protein is a recombinant protein.In some embodiments, the TSG-6 protein comprises a polypeptide havingthe sequence set forth in SEQ ID NO: 2 or a polypeptide having at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:2. In some embodiments, the TSG-6 protein used in the methods comprisesa polypeptide having the sequence set forth in any of SEQ ID NOS: 1-31or a species variant or allelic variant thereof. In some embodiments,the HC1 used in the methods comprises a polypeptide having the sequenceset forth in SEQ ID NO: 47 or a polypeptide having at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 47. In someembodiments, inter-α-inhibitor (IαI) protein used in the methods as asource of HC1 also comprises HC2 and bikunin linked by a chondroitinsulfate chain. In some embodiments, the HC1 comprises a polypeptidehaving the sequence set forth in any of SEQ ID NOS: 46-47 or a speciesvariant or allelic variant thereof. In some embodiments, HC2 comprises apolypeptide having the sequence set forth in SEQ ID NO: 49 or apolypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence amino acid identity to the polypeptide having the sequenceset forth in SEQ ID NO: 49. In some embodiments, the HC2 used in themethods comprises a polypeptide having the sequence set forth in any ofSEQ ID NOS: 48-49 or a species variant or allelic variant thereof. Insome embodiments, bikunin comprises a polypeptide having the sequenceset forth in SEQ ID NO: 53 or a polypeptide having at least 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 53. In someembodiments, the bikunin used in the methods comprises a polypeptidehaving the sequence set forth in any of SEQ ID NOS: 52-53 or a speciesvariant or allelic variant thereof. In some embodiments, the IαI proteinused in the methods is isolated from blood, serum, plasma, amnioticmembrane, chorionic membrane, amniotic fluid, or a combination thereof.In some embodiments, the IαI protein used in the methods is isolatedfrom serum. In some embodiments, the IαI protein used in the methods isisolated from human serum. In some embodiments, the IαI protein used inthe methods is produced by a mammalian cell. In some embodiments, thecell is a human cell. In some embodiments, the cell is an amnioticmembrane cell. In some embodiments, the cell is an umbilical cord cell.In some embodiments, the cell is an amniotic membrane cell from anumbilical cord. In some embodiments, the amniotic membrane cell is anamniotic epithelial cell. In some embodiments, the amniotic membranecell is an umbilical cord epithelial cell. In some embodiments, theamniotic membrane cell is an amniotic stromal cell. In some embodiments,the amniotic membrane cell is an umbilical cord stromal cell. In someembodiments, the IαI and TSG-6 protein are contacted at a molar ratio of1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. Insome embodiments, the IαI and TSG-6 protein are contacted at a molarratio of 3:1. In some embodiments of the method, the weight averagemolecular weight of the HMW HA is between about 500 kDa and about 10,000kDa, between about 800 kDa and about 8,500 kDa, between about 1100 kDaand about 5,000 kDa, or between about 1400 kDa and about 3,500 kDa. Insome embodiments of the method, the weight average molecular weight ofthe HMW HA is 3,000 kDa. In some embodiments, HMW HA is immobilized bydirect linkage to a solid support. In some embodiments of the method,HMW HA is immobilized by indirect linkage to a solid support. In someembodiments of the method, HMW HA is immobilized by covalent attachmentto the solid support. In some embodiments of the method, HMW HA isimmobilized by non-covalent attachment to the solid support. In someembodiments of the method, HMW HA is immobilized by linkage to a solidsupport via a cleavable linker. In some embodiments, the linker is achemically or enzymatically cleavable linker. In some embodiments, themethod further comprises dissociation of the rcHC-HA/PTX3 complex fromthe solid support following step (b). In some embodiments, dissociationcomprises cleavage of a cleavable linker. In some embodiments, themethod further comprises purification of the dissociated rcHC-HA/PTX3complex. In some embodiments, purification comprises affinitypurification, centrifugation, filtration, chromatography or acombination thereof. In some embodiments of the method, PTX3, IαI HC1 orTSG-6 polypeptides comprise an affinity tag. In some embodiments, theaffinity tag is selected from among a hemagglutinin tag, apoly-histidine tag, a myc tag, a FLAG tag, a glutathione-S-transferase(GST) tag. In some embodiments, HMW HA is immobilized by binding HMW HAto an intermediary polypeptide. In some embodiments, the intermediarypolypeptide is covalently attached to the solid support. In someembodiments, binding HMW HA to the intermediary polypeptide isnon-covalent. In some embodiments, the intermediary polypeptide is an HAbinding protein (HABP). In some embodiments, the intermediarypolypeptide is an HABP selected from among HAPLN1, HAPLN2, HAPLN3,HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44,stabilin-1, stabilin-2, or a portion thereof sufficient to bind HA. Insome embodiments, the intermediary polypeptide is versican. In someembodiments, the intermediary polypeptide comprises a link module. Insome embodiments, the intermediary polypeptide comprises a link moduleof HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan,brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2. In someembodiments, the intermediary polypeptide comprises a link module ofversican. In some embodiments, the intermediary polypeptide comprises apolypeptide set forth in any of SEQ ID NOS: 54-99. In some embodiments,the intermediary polypeptide comprises a polypeptide linker. In someembodiments, the linker is attached to the solid support. In someembodiments, the method further comprises dissociation of thercHC-HA/PTX3 complex from the intermediary polypeptide following step(b). In some embodiments, dissociation of the rcHC-HA/PTX3 complex fromthe intermediary polypeptide comprises contacting the complex with adissociation agent. In some embodiments, the dissociation agent isguanidine hydrochloride or urea. In some embodiments, the dissociationagent is about 4 M to about 8M guanidine hydrochloride. In someembodiments, the intermediary polypeptide or linker comprises aproteolytic cleavage sequence. In some embodiments, dissociation of thercHC-HA/PTX3 complex comprises cleaving the intermediary polypeptide orlinker at proteolytic cleavage sequence. In some embodiments, cleavingcomprises contacting the proteolytic cleavage sequence with a protease.In some embodiments, the protease is selected from among furin, 3Cprotease, caspase, matrix metalloproteinase and TEV protease.

Described herein, in certain embodiments, are methods of producing areconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprisingcontacting a PTX3/HA complex immobilized to a solid support withinter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) andTSG-6. In some embodiments, the PTX3/HA complex is produced bycontacting high molecular weight hyaluronan (HMW HA) with a pentraxin 3(PTX3) protein under conditions effective to form a complex of PTX3 andHMW HA (PTX3/HA), wherein the HMW HA is immobilized to a solid support.In some embodiments, the method further comprises removing unbound PTX3protein prior to contacting the PTX3/HA complex with IαI and TSG-6. Insome embodiments, the method further comprises removing unboundTSG-6. Insome embodiments, the PTX3 protein is a recombinant protein. In someembodiments, the PTX3 protein comprises a polypeptide having thesequence set forth in SEQ ID NO: 33 or a polypeptide having at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:33. In some embodiments, the PTX3 protein used in the methods is amultimeric protein. In some embodiments, the PTX3 protein used in themethods is a homomultimer. In some embodiments, PTX3 homomultimer is adimer, trimer, tetramer, pentamer, hexamer, octamer. In someembodiments, the PTX3 homomultimer is an octamer. In some embodiments,PTX3 comprises a modified multimerization domain or a heterogeneousmultimerization domain. In some embodiments, TSG-6 is a recombinantprotein. In some embodiments, TSG-6 comprises a polypeptide having thesequence set forth in SEQ ID NO: 2 or a polypeptide having at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identityto the polypeptide having the sequence set forth in SEQ ID NO: 2. Insome embodiments, HC1 comprises a polypeptide having the sequence setforth in SEQ ID NO: 47 or a polypeptide having at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 47. In someembodiments, IαI protein also comprises HC2. In some embodiments, HC2comprises a polypeptide having the sequence set forth in SEQ ID NO: 49or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% sequence amino acid identity to the polypeptide having thesequence set forth in SEQ ID NO: 49. In some embodiments, the IαIprotein also comprises bikunin. In some embodiments, bikunin comprises apolypeptide having the sequence set forth in SEQ ID NO: 53 or apolypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence amino acid identity to the polypeptide having the sequenceset forth in SEQ ID NO: 53. In some embodiments, IαI also comprises achondroitin sulfate chain. In some embodiments, the IαI protein is arecombinant protein. In some embodiments, the IαI protein is isolatedfrom blood, plasma, serum, amniotic membrane, chorionic membrane,amniotic fluid, or a combination thereof. In some embodiments, the IαIprotein is isolated from serum. In some embodiments, the IαI protein isisolated from human serum. In some embodiments, the IαI and TSG-6protein are contacted at a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. In some embodiments, the IαI andTSG-6 protein are contacted at a molar ratio of 3:1. In someembodiments, the weight average molecular weight of the HMW HA isbetween about 500 kDa and about 10,000 kDa, between about 800 kDa andabout 8,500 kDa, between about 1100 kDa and about 5,000 kDa, or betweenabout 1400 kDa and about 3,500 kDa. In some embodiments, the weightaverage molecular weight of the HMW HA is 3,000 kDa. In someembodiments, HMW HA is immobilized by direct linkage to a solid supportIn some embodiments, HMW HA is immobilized by indirect linkage to asolid support. In some embodiments, HMW HA is immobilized by covalentattachment to the solid support. In some embodiments, HMW HA isimmobilized by non-covalent attachment to the solid support. In someembodiments, the method further comprises dissociation of thercHC-HA/PTX3 complex from the solid support. In some embodiments, themethod further comprises purification of the dissociated rcHC-HA/PTX3complex. In some embodiments, purification comprises affinitypurification, centrifugation, filtration, chromatography or acombination thereof. In some embodiments, the PTX3, IαI HC1 or TSG-6polypeptides comprise an affinity tag. In some embodiments, the affinitytag is selected from among a hemagglutinin tag, a poly-histidine tag, amyc tag, a FLAG tag, a glutathione-S-transferase (GST) tag. In someembodiments, HMW HA is immobilized by binding HMW HA to an intermediarypolypeptide. In some embodiments, the intermediary polypeptide iscovalently attached to the solid support. In some embodiments, thebinding HMW HA to the intermediary polypeptide is non-covalent. In someembodiments, the intermediary polypeptide is an HA binding protein(HABP). In some embodiments, the intermediary polypeptide is an HABPselected from among HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican,neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, ora portion thereof sufficient to bind HA. In some embodiments, theintermediary polypeptide is versican. In some embodiments, theintermediary polypeptide comprises a link module. In some embodiments,the intermediary polypeptide comprises a link module of HAPLN1, HAPLN2,HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan,TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, theintermediary polypeptide comprises a link module of versican. In someembodiments, the intermediary polypeptide comprises a polypeptide setforth in any of SEQ ID NOS: 54-99. In some embodiments, the intermediarypolypeptide comprises a polypeptide linker. In some embodiments, thelinker is attached to the solid support. In some embodiments, the methodfurther comprises dissociation of the rcHC-HA/PTX3 complex from theintermediary polypeptide. In some embodiments, dissociation of thercHC-HA/PTX3 complex from the intermediary polypeptide comprisescontacting the complex with a dissociation agent. In some embodiments,the dissociation agent is guanidine hydrochloride or urea. In someembodiments, the dissociation agent is about 4 M to about 8 M guanidinehydrochloride. In some embodiments, the intermediary polypeptide orlinker comprises a proteolytic cleavage sequence. In some embodiments,dissociation comprises cleaving the intermediary polypeptide or linkerat proteolytic cleavage sequence. In some embodiments, cleavingcomprises contacting the proteolytic cleavage sequence with a protease.In some embodiments, the protease is selected from among furin, 3Cprotease, caspase, matrix metalloproteinase and TEV protease.

Described herein, in certain embodiments, are reconstituted HC-HA(rcHC-HA/PTX3) complexes produced by any of the methods provided hereinfor generating rcHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are reconstitutedHC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weighthyaluronan (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3complexes promote the M2 polarization of a macrophage. Described herein,in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complexescomprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1,wherein the nHC-HA/PTX3 complexes promote the M2 polarization of amacrophage. Described herein, in certain embodiments, are methods forinducing the M2 polarization of macrophages comprising contacting amacrophage with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are reconstitutedHC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weighthyaluronan (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3complexes reduce the expression of IL-12p40 in LPS-stimulatedmacrophages, wherein the level of IL-12p40 expressed by LPS-stimulatedmacrophages is lower when the LPS-stimulated macrophages are contactedthe rcHC-HA/PTX3 complexes compared to the level of IL-12p40 expressedby LPS-stimulated macrophages in the absence of the rcHC-HA/PTX3complexes. In some embodiments, the level of IL-12p40 mRNA is reduced.In some embodiments, the level of IL-12p40 protein is reduced.

Described herein, in certain embodiments, are nativeHC-HA/PTX3(nHC-HA/PTX3) complexes comprising high molecular weighthyaluronan (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3complexes reduce the expression of IL-12p40 in LPS-stimulatedmacrophages, wherein the level of IL-12p40 expressed by LPS-stimulatedmacrophages is lower when the LPS-stimulated macrophages are contactedthe nHC-HA/PTX3 complexes compared to the level of IL-12p40 expressed byLPS-stimulated macrophages in the absence of the nHC-HA/PTX3 complexes.In some embodiments, the level of IL-12p40 mRNA is reduced. In someembodiments, the level of IL-12p40 protein is reduced.

Described herein, in certain embodiments, are methods for reducing thelevel of IL-12p40 expressed by LPS-stimulated macrophages comprising,contacting an LPS-stimulated macrophages with an rcHC-HA/PTX3 orisolated nHC-HA/PTX3 complex. In some embodiments, the level of IL-12p40mRNA is reduced. In some embodiments, the level of IL-12p40 protein isreduced.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complexes reducethe expression of IL-12p70 protein in LPS-stimulated macrophages,wherein the amount of IL-12p70 protein expressed by LPS-stimulatedmacrophages is lower when the LPS-stimulated macrophages are contactedthe rcHC-HA/PTX3 complexes compared to the amount of IL-12p70 proteinexpressed by LPS-stimulated macrophages in the absence of thercHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complexes reduces theexpression of IL-12p70 protein in LPS-stimulated macrophages, whereinthe amount of IL-12p70 protein expressed by LPS-stimulated macrophagesis lower when the LPS-stimulated macrophages are contacted thenHC-HA/PTX3 complexes compared to the amount of IL-12p70 proteinexpressed by LPS-stimulated macrophages in the absence of thenHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are methods for reducing thelevel of IL-12p70 protein expressed by LPS-stimulated macrophagescomprising, contacting an LPS-stimulated macrophages with anrcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complexes reducethe expression of IL-23 in LPS-stimulated macrophages, wherein the levelof IL-23 expressed by LPS-stimulated macrophages is lower when theLPS-stimulated macrophages are contacted the rcHC-HA/PTX3 complexescompared to the level of IL-23 expressed by LPS-stimulated macrophagesin the absence of the rcHC-HA/PTX3 complexes. In some embodiments, thelevel of IL-23 mRNA is reduced. In some embodiments, the level of IL-23protein is reduced.

Described herein, in certain embodiments, are native HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the complexes reduces theexpression of IL-23 in LPS-stimulated macrophages, wherein the level ofIL-23 expressed by LPS-stimulated macrophages is lower when theLPS-stimulated macrophages are contacted the nHC-HA/PTX3 complexescompared to the level of IL-23 expressed by LPS-stimulated macrophagesin the absence of the nHC-HA/PTX3 complex. In some embodiments, thelevel of IL-23 mRNA is reduced. In some embodiments, the level of IL-23protein is reduced.

Described herein, in certain embodiments, are methods for reducing thelevel of IL-23 expressed by LPS-stimulated macrophages comprising,contacting an LPS-stimulated macrophages with an rcHC-HA/PTX3 orisolated nHC-HA/PTX3 complex. In some embodiments, the level of IL-23mRNA is reduced. In some embodiments, the level of IL-23 protein isreduced.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complexes increasethe expression of IL-10 in LPS-stimulated macrophages, wherein the levelof IL-10 expressed by LPS-stimulated macrophages is higher when theLPS-stimulated macrophages are contacted the rcHC-HA/PTX3 complexescompared to the level of IL-10 expressed by LPS-stimulated macrophagesin the absence of the rcHC-HA/PTX3 complexes. In some embodiments, thelevel of IL-10 mRNA is increased. In some embodiments, the level ofIL-10 protein is increased.

Described herein, in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, wherein the complexes increase the expression ofIL-10 in LPS/IFNγ-stimulated macrophages, wherein the amount of IL-10expressed by LPS/IFNγ -stimulated macrophages is higher when theLPS-stimulated macrophages are contacted the nHC-HA/PTX3 complexescompared to the amount of IL-10 expressed by LPS/IFNγ -stimulatedmacrophages in the absence of the nHC-HA/PTX3 complexes. In someembodiments, the level of IL-10 mRNA is increased. In some embodiments,the level of IL-10 protein is increased.

Described herein, in certain embodiments, are methods for increasing thelevel of IL-10 expressed by LPS-stimulated macrophages comprising,contacting an LPS-stimulated macrophages with an rcHC-HA/PTX3 orisolated nHC-HA/PTX3 complex. In some embodiments, the level of IL-10mRNA is increased. In some embodiments, the level of IL-10 protein isincreased.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC -HA/PTX3complexes promoteapoptosis of LPS-stimulated neutrophils but do not promote apoptosis inresting neutrophils. In some embodiments, a reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complex, comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, promotes apoptosis of LPS-stimulated neutrophilswherein the number of LPS-stimulated neutrophils that are apoptotic in asample of LPS-stimulated neutrophils is higher when the sample iscontacted the rcHC-HA/PTX3 complex compared to the number ofLPS-stimulated neutrophils that are apoptotic in the sample in theabsence of the rcHC-HA/PTX3 complex.

Described herein, in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complex comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complexes promoteapoptosis of LPS-stimulated neutrophils but do not promote apoptosis inresting neutrophils. In some embodiments, a nHC-HA/PTX3 complex,comprising high molecular weight hyaluronan (HMW HA), PTX3, and IαI HC1,promotes apoptosis of LPS-stimulated neutrophils wherein the number ofLPS-stimulated neutrophils that are apoptotic in a sample ofLPS-stimulated neutrophils is higher when the sample is contacted thenHC-HA/PTX3 complex compared to the number of LPS-stimulated neutrophilsthat are apoptotic in the sample in the absence of the nHC-HA/PTX3complex.

Described herein, in certain embodiments, are methods for inducingapoptosis of LPS-stimulated neutrophils comprising, contacting anLPS-stimulated neutrophil with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3complex.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3 complexes promotephagocytosis of apoptotic neutrophils, wherein the number of apoptoticneutrophils that are phagocytosed by LPS-stimulated macrophages in asample of apoptotic neutrophils and LPS-stimulated macrophages is higherwhen the sample is contacted with the rcHC-HA/PTX3 complexes compared tothe number of neutrophils that are phagocytosed by LPS-stimulatedmacrophages in the sample in the absence of the rcHC-HA/PTX3 complexes.Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the complexes promote phagocytosisof apoptotic neutrophils, wherein the number of apoptotic neutrophilsthat are phagocytosed by resting macrophages in a sample of apoptoticneutrophils and resting macrophages is higher when the sample iscontacted with the rcHC-HA/PTX3 complexes compared to the number ofneutrophils that are phagocytosed by resting macrophages in the samplein the absence of the rcHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complexes promotesphagocytosis of apoptotic neutrophils, wherein the number of apoptoticneutrophils that are phagocytosed by LPS-stimulated macrophages in asample of apoptotic neutrophils and LPS-stimulated macrophages is higherwhen the sample is contacted with the nHC-HA/PTX3 complexes compared tothe number of neutrophils that are phagocytosed by LPS-stimulatedmacrophages in the sample in the absence of the nHC-HA/PTX3 complexes.Described herein, in certain embodiments, are native HC-HA/PTX3(nHC-HA/PTX3) complexes comprising high molecular weight hyaluronan (HMWHA), PTX3, and IαI HC1, wherein the nHC-HA/PTX3 complexes promotesphagocytosis of apoptotic neutrophils, wherein the number of apoptoticneutrophils that are phagocytosed by resting macrophages in a sample ofapoptotic neutrophils and LPS-stimulated macrophages is higher when thesample is contacted with the nHC-HA/PTX3 complexes compared to thenumber of neutrophils that are phagocytosed by resting macrophages inthe sample in the absence of the nHC-HA/PTX3 complexes.

Described herein, in certain embodiments, are methods inducingphagocytosis of apoptotic neutrophils comprising, contacting a samplecontaining apoptotic neutrophils and LPS-stimulated or restingmacrophages with an rcHC-HA/PTX3 or isolated nHC-HA/PTX3 complex.

Described herein, in certain embodiments, are reconstituted HC-HA/PTX3(rcHC-HA/PTX3) complexes comprising high molecular weight hyaluronan(HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA/PTX3complexes promoteattachment of LPS-stimulated macrophages to at least the same level as anative HC-HA/PTX3 (nHC-HA/PTX3) complex isolated from human umbilicalcord, human amniotic membrane, or a combination of nHC-HA/PTX3 complexesfrom both human umbilical cord and human amniotic membrane, whereinattachment comprises the contacting LPS-stimulated macrophages to thercHC-HA/PTX3 or nHC-HA/PTX3 complexes immobilized to a solid support. Insome embodiments, the nHC-HA/PTX3 is isolated from human umbilical cord.In some embodiments, the nHC-HA/PTX3 is isolated from human amnioticmembrane. In some embodiments, the nHC-HA/PTX3 is isolated from acombination of nHC-HA/PTX3 complexes from both human umbilical cord andhuman amniotic membrane

In some embodiments, the weight average molecular weight of the HMW HAof the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is between about 500 kDa andabout 10,000 kDa, between about 800 kDa and about 8,500 kDa, betweenabout 1100 kDa and about 5,000 kDa, or between about 1400 kDa and about3,500 kDa. In some embodiments, contains, the weight average molecularweight of the HMW HA of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is about3,000 kDa.

In some embodiments, HC1 of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex iscovalently linked to HA.

In some embodiments, the PTX3 protein of the rcHC-HA/PTX3 complex is arecombinant protein. In some embodiments, PTX3 of the rcHC-HA/PTX3complex comprises a polypeptide having the sequence set forth in SEQ IDNO: 33 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence amino acid identity to the polypeptide havingthe sequence set forth in SEQ ID NO: 33. In some embodiments, the PTX3protein used in the methods is a multimeric protein. In someembodiments, the PTX3 protein used in the methods is a homomultimer. Insome embodiments, the PTX3 homomultimer is a dimer, trimer, tetramer,pentamer, hexamer, or octamer. In some embodiments, the PTX3homomultimer is a trimer, tetramer, or octamer. In some embodiments, thePTX3 homomultimer is an octamer.

In some embodiments, the IαI HC1 of the rcHC-HA/PTX3 complex comprises apolypeptide having the sequence set forth in SEQ ID NO: 47 or apolypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or99% sequence amino acid identity to the polypeptide having the sequenceset forth in SEQ ID NO: 47. In some embodiments, the IαI HC1 of thercHC-HA/PTX3 complex is a recombinant protein.

In some embodiments, the rcHC-HA/PTX3 complex comprises TSG-6. In someembodiments, the TSG-6 protein is a recombinant protein. In someembodiments, the TSG-6 protein comprises a polypeptide having thesequence set forth in SEQ ID NO: 2 or a polypeptide having at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identityto the polypeptide having the sequence set forth in SEQ ID NO: 2.

In some embodiments, the PTX3, IαI HC1 or TSG-6 polypeptides of thercHC-HA/PTX3 complex comprise an affinity tag. In some embodiments, theaffinity tag is selected from among is selected from among ahemagglutinin tag, a poly-histidine tag, a myc tag, a FLAG tag, aglutathione-S-transferase (GST) tag.

Described herein, in certain embodiments, is a pharmaceuticalcomposition, comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex describedherein or produced by the methods provided herein. In some embodiments,pharmaceutical composition further comprises a pharmaceuticallyacceptable carrier or excipient. In some embodiments, pharmaceuticalcomposition is in the form of a solution, suspension, powder, ointment,tablet, capsule, or an aerosol. In some embodiments, pharmaceuticalcomposition is in the form of a solid, a cross-linked gel, or aliposome. In some embodiments, pharmaceutical composition is in the formof a cross-linked hyaluronan hydrogel. In some embodiments,pharmaceutical composition comprises a natural polymer. In someembodiments, natural polymer comprises fibronectin, collagen, laminin,keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate,chondroitin sulfate, or combinations thereof. In some embodiments,pharmaceutical composition further comprises an anti-inflammatory agent,an anti-scarring agent, an anti-neoplastic agent, a chemotherapeuticagent, an immunosuppressive agent, a cytotoxic agent, an antimicrobialagent, or a combination thereof.

Described herein, in certain embodiments, is a use of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein for the production of a medicament.

Described herein, in certain embodiments, is a combination comprising:(a) an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or producedby the methods provided herein; and (b) an anti-inflammatory agent, ananti-scarring agent, an anti-neoplastic agent, a chemotherapeutic agent,an immunosuppressive agent, a cytotoxic agent, an antimicrobial agent ora combination thereof.

Described herein, in certain embodiments, are methods of treatment,comprising administering a pharmaceutical composition comprising annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein.

Described herein, in certain embodiments, are methods of preventing orreversing scar formation or fibrosis in a tissue, comprisingadministering to the subject in need thereof an effective amount of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein. In some embodiments, the method comprisescontacting the tissue with an effective amount of the nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, the scar is a dermatitisscar, a keloid scar, contracture scar, a hypertrophic scar, or a scarresulting from acne. Described herein, in certain embodiments, is a useof an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or producedby the methods provided herein to reduce or prevent scarring. In someembodiments, administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein reduces orprevents scarring or fibrosis by decreasing or inhibiting TGF-βsignaling in the tissue.

Described herein, in certain embodiments, are methods of preventing orreducing inflammation in a subject in need thereof, comprisingadministering to the subject an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the method comprises contactinginflamed tissues with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In someembodiments, the inflammation is acute inflammation or chronicinflammation. In some embodiments, the subject has an inflammatorydisorder. In some embodiments, the inflammatory disorder is amacrophage-mediated inflammatory disorder, a Th-17-mediated immunedisorder or a T-cell mediated inflammatory disorder. In someembodiments, the subject has an autoimmune disorder, an allergy, aleukocyte defect, an infection, graft versus host disease, tissuetransplant rejection, or combinations thereof. In some embodiments, theinflammatory disorder is rheumatoid arthritis. In some embodiments, theinflammatory disorder is an inflammatory disorder of the eye. In someembodiments, the inflammatory disorder is conjunctivitis, keratitis,blepharitis, blepharoconjunctivitis, scleritis, episcleritis, uveitis,retinitis, or choroiditis. In some embodiments, the acute inflammationis caused by myocardial infarction, stroke, endotoxin shock or sepsis.In some embodiments, the subject has atherosclerosis. In someembodiments, the subject has cancer. In some embodiments, the subjecthas inflammation of a solid tumor. In some embodiments, the subject isadministered the nHC-HA/PTX3 or rcHC-HA/PTX3 complex in combination withan additional anti-inflammatory agent. In some embodiments, theadditional anti-inflammatory agent is selected from among an anti-TGF-βantibody, an anti-TGF-β receptor blocking antibody, an anti-TNFantibody, an anti-TNF receptor blocking antibody, an anti-IL1β antibody,an anti-IL1β receptor blocking antibody, an anti-IL-2 antibody, ananti-IL-2 receptor blocking antibody, an anti-IL-6 antibody, ananti-IL-6 receptor blocking antibody, an anti IL-12 antibody, an antiIL-12 receptor blocking antibody, an anti-IL-17 antibody, anti-IL-17receptor blocking antibody, an anti-IL-23 antibody, or an anti-IL-23receptor blocking antibody. In some embodiments, the Type 1 interferonis IFN-α or IFN-β. Described herein, in certain embodiments, is a use ofan nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced bythe methods provided herein to reduce or prevent inflammation.

Described herein, in certain embodiments, are methods of treating a skinwound or ulcer in a subject in need thereof, comprising administering tothe subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein or produced by the methods provided herein. Insome embodiments, the methods comprise contacting the skin wound orulcer with an effective amount of the nHC-HA/PTX3 or rcHC-HA/PTX3complex. Described herein, in certain embodiments, is a use of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein to treat a skin wound or ulcer. In someembodiments, the skin wound or ulcer is a non-healing ulcer.

Described herein, in certain embodiments, are methods of promoting orinducing bone formation in a subject in need thereof, comprisingadministering to the administering to the subject an effective amount ofthe rcHC-HA/PTX3 complex or nHC-HA/PTX3 complex described herein orproduced by the methods provided herein. In some embodiments, thesubject has arthritis, osteoporosis, alveolar bone degradation, Paget’sdisease, or a bone tumor. Described herein, in certain embodiments, is ause of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein orproduced by the methods provided herein to promote or induce boneformation in a subject.

Described herein, in certain embodiments, are methods of preventing orreducing abnormal angiogenesis in a subject in need thereof, comprisingadministering to the subject an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the subject has wet age-relatedmacular degeneration (wARMD) or diabetic proliferative retinopathy. Insome embodiments, the subject has cancer. In some embodiments, thesubject has a solid tumor. In some embodiments, the subject isadministered the nHC-HA/PTX3 or rcHC-HA/PTX3 complex in combination withan anti-cancer therapy. In some embodiments, the anticancer therapycomprises administration of an anti-neoplastic agent, a cytotoxic agent,an anti-angiogenic agent, a chemotherapeutic agent, or radiationtherapy. In some embodiments, the anticancer therapy is administeredsequentially, concurrently or intermittently with the nHC-HA/PTX3 orrcHC-HA/PTX3 complex. Described herein, in certain embodiments, is a useof an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or producedby the methods provided herein to reduce or prevent angiogenesis.

Described herein, in certain embodiments, are methods of preventingtransplant rejection in a transplant recipient, comprising administeringto the transplant recipient an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the method comprise contacting thetransplant with the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administeredbefore a transplantation procedure, after a transplantation procedure,or during a transplantation procedure. In some embodiments, thenHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered in combination withan immunosuppressive agent. Described herein, in certain embodiments, isa use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein orproduced by the methods provided herein to prevent transplant rejectionin a transplant recipient. In some embodiments, the transplant is acorneal transplant.

Described herein, in certain embodiments, are methods of inducing stemcell expansion in a subject in need thereof, comprising administering tothe subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein or produced by the methods provided herein. Insome embodiments, the method comprise contacting the stem cell with thenHC-HA/PTX3 or rcHC-HA/PTX3 complex. Described herein, in certainembodiments, is a use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein to inducestem cell expansion. In some embodiments, administration of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein induces stem cell expansion by suppression ofTGF-β signaling and/or upregulation of BMP signaling pathways. In someembodiments, administration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein induces stemcell expansion by reprogramming differentiated cells into stem cells (orinduced progenitor cells, iPSCs).

Described herein, in certain embodiments, is a use of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein for cell therapy.

Described herein, in certain embodiments, are methods of cell therapy ina subject in need thereof, comprising administering to the subject acomposition comprising an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein in combination with a therapeutic cell. In someembodiments, the therapeutic cell and the nHC-HA/PTX3 or rcHC-HA/PTX3complex are administered locally to a damaged tissue. In someembodiments, the therapeutic cell and the nHC-HA/PTX3 or rcHC-HA/PTX3complex are administered systemically. In some embodiments, thetherapeutic cell is a stem cell. In some embodiments, the therapeutic isa stem cell. In some embodiments, the cell therapy comprisesadministration of a stem cell. In some embodiments, the stem cell is amesenchymal stem cell. In some embodiments, the stem cells are inducedprogenitor stem cells. In some embodiments, the cell therapy comprisesadministration of differentiated cells. In some embodiments, therapeuticcell is an insulin producing cell. In some embodiments, the insulinproducing cell is an islet cell. In some embodiments, the subject hasdiabetes mellitus type 1.

Described herein, in certain embodiments, are methods of cell therapy ina subject in need thereof, comprising administering to the administeringto the subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein or produced by the methods provided herein incombination with a cell contained in a cell delivery device. In someembodiments, the cell is contained in a microcapsule. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to themicrocapsule. In some embodiments, the microcapsule is administeredlocally to a damaged tissue. In some embodiments, the therapeutic celland the nHC-HA/PTX3 or rcHC-HA/PTX3 complex are administeredsystemically. In some embodiments, the therapeutic cell is a stem cell.In some embodiments, the therapeutic is a stem cell. In someembodiments, the cell therapy comprises administration of a stem cell.In some embodiments, the stem cell is a mesenchymal stem cell. In someembodiments, the stem cells are induced progenitor stem cells. In someembodiments, the cell therapy comprises administration of differentiatedcells. In some embodiments, therapeutic cell is an insulin producingcell. In some embodiments, the insulin producing cell is an islet cell.In some embodiments, the subject has diabetes mellitus type 1.

Described herein, in certain embodiments, are methods of preventing orreducing pain in a subject in need thereof, comprising administering tothe subject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein or produced by the methods provided herein,wherein the pain is caused by chemical bum, severe bacterial keratitis,Stevens-Johnson syndrome, toxic epidermal necrolysis, irradiation ofocular tumors. Described herein, in certain embodiments, is a use of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein to reduce pain in a subject, wherein the pain iscaused by chemical burn, severe bacterial keratitis, Stevens-Johnsonsyndrome, toxic epidermal necrolysis, irradiation of ocular tumors.

Described herein, in certain embodiments, are methods of inducing orpromoting tissue regeneration in a subject in need thereof, comprisingadministering to the subject an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the methods comprise contactingdamaged tissue of the subject with an effective amount of thenHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, the tissue isbone or gum, corneal tissue, or conjunctival tissue. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered incombination with a therapeutic cell, a plurality of therapeutic cells ora tissue transplant. In some embodiments, the tissue transplant is anallograft or an autograft. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complex is administered in combination with a tissue basedtherapy. Described herein, in certain embodiments, is a use of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein to induce or promote tissue regeneration in asubject.

Described herein, in certain embodiments, are methods of treatingfibrosis in a subject in need thereof, comprising administering to thesubject an effective amount of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein. In someembodiments, the treatment inhibits or prevents scarring. Describedherein, in certain embodiments, is a use of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein to treat fibrosis in a subject.

Described herein, in certain embodiments, are methods of treatingobesity or insulin resistance in a subject in need thereof, comprisingadministering to the subject an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the treatment inhibits ordecreases the amount of M1 adipose tissue macrophages in the subject.Described herein, in certain embodiments, are methods of inhibiting ordecreasing the amount of M1 adipose tissue macrophages in a subject inneed thereof, administering to the subject an effective amount of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein. In some embodiments, the subject has beendiagnosed with obesity or insulin-resistance. Described herein, incertain embodiments, is a use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein to treatobesity or insulin resistance in a subject.

Described herein, in certain embodiments, are methods of treatingconjunctivochalasis in a subject in need thereof, comprisingadministering to the subject an effective amount of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein or produced by the methodsprovided herein. In some embodiments, the methods comprise contactingthe conjunctiva of the subject with an effective amount of thenHC-HA/PTX3 or rcHC-HA/PTX3 complex. Described herein, in certainembodiments, is a use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdescribed herein or produced by the methods provided herein to treatconjunctivochalasis in a subject.

Described herein, in certain embodiments, are cell cultures comprising asubstrate suitable for culturing a cell and a rcHA/PTX3 complex or thenHC-HA/PTX3 complex described herein or produced by the methods providedherein, In some embodiments, the rcHA/PTX3 complex or the nHC-HA/PTX3complex immobilized to the substrate.

Described herein, in certain embodiments, are methods of treatment,wherein the subject is administered an effective amount of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein in combination with an additional therapeuticagent. In some embodiments, the additional therapeutic agent is selectedfrom an interferon, an anti-tumor necrosis agent, an interleukin-1(IL-1) receptor antagonist, an interleukin-2 (IL-2) receptor antagonist,an interleukin-6 (IL-6) receptor antagonist, an interleukin-12 (IL-12)receptor antagonist, an interleukin-17 (IL-17) receptor antagonist, aninterleukin-23 (IL-23) receptor antagonist, a cytotoxic agent, anantimicrobial agent, an interleukin, an immunomodulatory agent, anantibiotic, a T-cell co-stimulatory blocker, a disorder-modifyinganti-rheumatic agent, an immunosuppressive agent, an anti-lymphocyteantibody, an anti-angiogenesis agent, a chemotherapeutic agent, ananti-neoplastic agent, an anti-metabolite, an Akt inhibitor, an IGF-1inhibitor, an angiotensin II antagonist, a cyclooxygenase inhibitor, anheparanase inhibitor, a lymphokine inhibitor, a cytokine inhibitor, anIKK inhibitor, a P38MAPK inhibitor, an anti-apoptotic pathway inhibitor,an apoptotic pathway agonist, a PPAR agonist, an inhibitors of Ras, atelomerase inhibitor, a protease inhibitor, a metalloproteinaseinhibitor, an aminopeptidase inhibitor, a SHIP activator andcombinations thereof. In some embodiments, the antimicrobial agent is anantiviral, antibacterial or antifungal agent.

Described herein, in certain embodiments, are methods of treatment,wherein the subject is administered an effective amount of annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein and the subject is a mammal. In someembodiments, the mammal is a human.

Described herein, in certain embodiments, are methods of treatment,wherein the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a solidsurface. In some embodiments, the solid surface is a surface or aportion thereof of a nanoparticle, a bead, a microcapsule or implantablemedical device.

Described herein, in certain embodiments, is are medical devices,comprising a substrate coated with an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein or produced by the methods provided herein. Insome embodiments, the substrate comprises at least one of a stent, ajoint, a screw, a rod, a pin, a plate, a staple, a shunt, a clamp, aclip, a suture, a suture anchor, an electrode, a catheter, a lead, agraft, a dressing, a pacemaker, a pacemaker housing, a cardioverter, acardioverter housing, a defibrillator, a defibrillator housing, aprostheses, an ear drainage tube, an ophthalmic implant, an orthopedicdevice, a vertebral disk, a bone substitute, an anastomotic device, aperivascular wrap, a colostomy bag attachment device, a hemostaticbarrier, a vascular implant, a vascular support, a tissue adhesive, atissue sealant, a tissue scaffold, and an intraluminal device.

Described herein, in certain embodiments, is a device comprising annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein immobilized on a surface. In some embodiments,the surface is a polystyrene, polyethylene, silica, metallic orpolymeric surface. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complex is attached to a microcapsule. In some embodiments, thenHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a nanoparticle. Insome embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached toa bead, a chip, a glass slide, or a filter. In some embodiments, thenHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached to a contact lens. Insome embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is attached toa surgical implant or prosthesis. In some embodiments, the implant orprosthesis is an artificial joint, a bone implant, a suture, or a stent.In some embodiments, the artificial joint is an artificial hip joint, anartificial knee, an artificial glenohumeral joint, or an artificialknee. In some embodiments, the stent is a coronary stent, a ureteralstent, a urethral stent, a prostatic stent, esophageal stent, or a bonestent.

Described herein, in certain embodiments, is are medical devicescomprising: a structure adapted for implantation into a patient, whereina surface or a portion of a surface of the structure is attached tocomprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein orproduced by the methods provided herein. In some embodiments, attachmentcomprises covalent or non-covalent attachment of the nHC-HA/PTX3 orrcHC-HA/PTX3 complex to the surface or portion of a surface of thestructure In some embodiments, attachment comprises coating the surfaceor a portion of a surface of the structure with a composition containingthe nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, thestructure is a vascular stent, an artificial joint, a suture, or amicrocapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complex inhibits the formation of a bacterial biofilm. In someembodiments, the microcapsule contains a therapeutic cell. In someembodiments, the therapeutic cell is a stem cell.

Described herein, in certain embodiments, are methods for modulatingmacrophage activity comprising contacting a macrophage with annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein in an amount sufficient to reduce or inhibit theexpression of IL-12 or IL-23 but also promote the expression of IL-10 inpolarizing macrophages from M1 to M2 phenotype. In some embodiments, themacrophage has been stimulated with a pro-inflammatory mediator. In someembodiments, the pro-inflammatory mediator is lipopolysaccharide (LPS),tumor necrosis factor α (TNF-α), interferon-gamma (IFN γ) or acombination thereof. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complex contacts the macrophage in vivo in a subject. Insome embodiments, the subject is a mammal. In some embodiments, themammal is a human. In some embodiments, the method is performed invitro. Described herein, in certain embodiments, are methods oftreatment comprising administration of macrophages that have beenmodulated by the method for modulating macrophage activity providedherein.

Described herein, in certain embodiments, is a kit, comprising annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein or produced by themethods provided herein, a device for administration of the compositionand, optionally, instructions for administration.

Described herein, in certain embodiments, is a combination or mixturecomprising: (a) a complex of PTX3 pre-bound to HMW HA (PTX3/HA); (b) aninter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1); and (c)TSG-6.

Described herein, in certain embodiments, is a combination or a mixturecomprising: (a) a complex of TSG-6 pre-bound to HC-HA; and (b) PTX3.

Described herein, in certain embodiments, are methods of producing areconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprising:(a) contacting (i) high molecular weight hyaluronan (HMW HA) immobilizedto a solid support, (ii) an inter-α-inhibitor (IαI) protein comprisingheavy chain 1 (HCl) and (iii) TSG-6 to form an rcHC-HA complex pre-boundto TSG-6; and (b) contacting rcHC-HA complex pre-bound to TSG-6 with apentraxin 3 (PTX3) protein to form an rcHC-HA/PTX3 complex.

Described herein, in certain embodiments, are methods of producing areconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex in vitro, comprisingcontacting (i) high molecular weight hyaluronan (HMW HA) immobilized toa solid support, (ii) pentraxin 3 (PTX3) protein, (iii)inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HCl) and (iv)Tumor necrosis factor αstimulated gene 6 (TSG-6) to form an immobilizedrcHC-HA/PTX3 complex.

Described herein, in certain embodiments, is a complex comprisingimmobilized HA bound to PTX3.

Described herein, in certain embodiments, are methods of producing acomplex comprising immobilized HA bound to PTX3 in vitro, comprisingcontacting high molecular weight hyaluronan (HMW HA) with a PTX3 proteinunder conditions effective to form a complex of PTX3 and HMW HA(PTX3/HA), wherein the HMW HA is immobilized to a solid support. In someembodiments, the PTX3 protein is a native PTX3 protein isolated from acell. In some embodiments, the cell is a mammalian cell. In someembodiments, the cell is a human cell. In some embodiments, the cell isan amniotic membrane cell. In some embodiments, the cell is an umbilicalcord cell. In some embodiments, the cell is an amniotic membrane cellfrom an umbilical cord. In some embodiments, the amniotic membrane cellis an amniotic epithelial cell. In some embodiments, the amnioticmembrane cell is an umbilical cord epithelial cell. In some embodiments,the amniotic membrane cell is an amniotic stromal cell. In someembodiments, the amniotic membrane cell is an umbilical cord stromalcell. In some embodiments, the PTX3 protein is a recombinant protein. Insome embodiments, the PTX3 protein comprises a polypeptide having thesequence set forth in SEQ ID NO: 2 or a polypeptide having at least 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identityto the polypeptide having the sequence set forth in SEQ ID NO: 2. Insome embodiments, the PTX3 protein used in the methods is a multimericprotein. In some embodiments, the PTX3 protein used in the methods is ahomomultimer. In some embodiments, the PTX3 homomultimer is a dimer,trimer, tetramer, pentamer, hexamer, octamer. In some embodiments, thePTX3 homomultimer is a trimer, tetramer, or octamer. In someembodiments, the PTX3 homomultimer is an octamer. In some embodiments,the PTX3 comprises a modified multimerization domain or a heterogeneousmultimerization domain. In some embodiments, the immobilizing HMW HAcomprises non-covalent attachment to the solid support. In someembodiments, the immobilizing HMW HA comprises binding HMW HA to anintermediary polypeptide. In some embodiments, the intermediarypolypeptide is covalently attached to the solid support. In someembodiments, binding HMW HA to the intermediary polypeptide isnon-covalent. In some embodiments, the intermediary polypeptide is an HAbinding protein (HABP). In some embodiments, the intermediarypolypeptide is an HABP selected from among HAPLN1, HAPLN2, HAPLN3,HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan, TSG-6, CD44,stabilin-1, stabilin-2, or a portion thereof sufficient to bind HA. Insome embodiments, the intermediary polypeptide is versican. In someembodiments, the intermediary polypeptide comprises a link module. Insome embodiments, the intermediary polypeptide comprises a link moduleof HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican, neurocan,brevican, phosphacan, TSG-6, CD44, stabilin-1, or stabilin-2. In someembodiments, the intermediary polypeptide comprises a link module ofversican. In some embodiments, the intermediary polypeptide comprises apolypeptide set forth in any of SEQ ID NOS: 54-99. Described herein, incertain embodiments, is a PTX3/HA complex produced by the precedingmethod. Described herein, in certain embodiments, is a pharmaceuticalcomposition, comprising the PTX3/HA complex produced by the precedingmethod. Described herein, in certain embodiments, is a use of thePTX3/HA complex for the production of a medicament. Described herein, incertain embodiments, are methods of treatment comprising administrationof the PTX3/HA complex for the prevention or inhibition of scarring,inflammation, angiogenesis, cancer, diabetes, obesity, or fibrosis.

Described herein, in certain embodiments, are methods for inducing ormaintaining pluripotency in a cell, comprising culturing the cell withan nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex, thereby inducing ormaintaining pluripotency in a cell. In some embodiments, the cellheterogeneously expresses a protein selected from among Sox2, myc, Oct4and KLF4. In some embodiments, the cell heterogeneously expresses one,two, or three proteins selected from among Sox2, myc, Oct4 and KLF4. Insome embodiments, the nHC-HA/PTX3 complex or rcHC-HA/PTX3 complex isimmobilized. In some embodiments, the cell is an adult differentiatedcell. In some embodiments, the cell is a fibroblast. In someembodiments, the cell is a human corneal fibroblast. In someembodiments, the cell is an embryonic stem cell, an adult stem cell, afetal stem cell, or an induced pluripotent stem cell. In someembodiments, the cell is a limbal epithelial progenitor cell, a limbalstromal niche cell, an umbilical cord stem cell, an amniotic membranestem cell or an adipose stem cell. In some embodiments, the nHC-HA/PTX3complex is an amniotic membrane nHC-HA/PTX3 complex. In someembodiments, the nHC-HA/PTX3 is an umbilical cord nHC-HA/PTX3 complex.In some embodiments, the methods further comprise purifying thenHC-HA/PTX3 complex by performing ultracentrifugation on an amnioticmembrane extract. In some embodiments, the methods further comprisepurifying the nHC-HA/PTX3 complex by performing ultracentrifugation onan amniotic membrane extract prepared in a PBS buffer to produce anHC-HA/PTX3 extract (i.e. nHC-HA/PTX3 soluble). In some embodiments, themethods further comprise purifying the nHC-HA/PTX3 complex by performingultracentrifugation on an amniotic membrane extract prepared in a GnHClbuffer to produce an nHC-HA/PTX3 extract (i.e. nHC-HA/PTX3 soluble). Insome embodiments, the methods further comprise purifying the nHC-HA/PTX3complex by performing ultracentrifugation on an umbilical cord extract.In some embodiments, the umbilical cord extract comprises umbilical cordamniotic membrane, umbilical cord stroma, Wharton’s jelly, or anycombination thereof. In some embodiments, the methods further comprisepurifying the nHC-HA/PTX3 complex by performing ultracentrifugation onan umbilical cord extract prepared in a PBS buffer to produce anHC-HA/PTX3 extract (i.e. nHC-HA/PTX3 soluble). In some embodiments, themethods further comprise purifying the nHC-HA/PTX3 complex by performingultracentrifugation on an umbilical cord extract prepared in a GnHClbuffer to produce an nHC-HA/PTX3 extract (i.e. nHC-HA/PTX3 soluble). Insome embodiments, the methods further comprise performing two, three orfour rounds of ultracentifugation. In some embodiments, the methodsfurther comprise performing four rounds of ultracentifugation. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises PTX3. Insome embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises asmall leucine rich proteoglycan (SLRP). In some embodiments, thenHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises PTX3 and a small leucinerich proteoglycan (SLRP). In some embodiments, the small leucine-richproteoglycan is selected from among decorin, biglycan, fibromodulin,lumican, PRELP (proline arginine rich end leucine-rich protein),keratocan, osteoadherin, epipycan, and osteoglycin. In some embodiments,the small leucine-rich proteoglycan is covalently attached to aglycosaminoglycan. In some embodiments, the glycosaminoglycan is keratansulfate.

It is to be understood that the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of any subject matter claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-M exemplifies purification of native HC-HA/PTX3 (nHC-HA/PTX3)complexes from amniotic membrane extract (AME) and analysis of proteincomposition and the size of HA. (A-B) Total protein and HAconcentrations in fractions obtained by CsCl/4 M guanidine HClultracentifugation. (C) HA stained by Stains-all dye in 0.5% agarosegel. (D-J) Analysis of proteins present in nHC-HA/PTX3 by Western blotusing antibodies against HCl (D and F), PTX3 (E and G), HC2 (H), HC3(I), bikunin (J), TSG-6 (K) or TSP-1 (L). NaOH or N = Treatment with0.05 N NaOH at 25° C. for 1 h. HAase or H = Treatment with 20 units/mlHAase at 60° C. for 2 h (F-K). Bar graph of relative protein amount (M)determined by dot assays using antibodies against various antigensincluding IGFBP 1-3, PF4 or TIMP-1.

FIGS. 2A-C exemplifies CD44 and TLR4 receptors mediate the attachment ofLPS-Stimulated macrophages to immobilized nHC-HA/PTX3. (A) Cellattachment. RAW264.7 cells (100 µl of 2.5x10⁵cells/ml) were seeded inimmobilized HA (2 µg/well) or nHC-HA/PTX3 (2 µg/well) (n=3) andstimulated with LPS (1 µg/ml). After incubation for 90 min, unattachedcells were removed and attached cells were counted by the CyQuant assay.The scale bar represents 100 µm. An asterisk (∗) indicates p values <0.05 (HA or nHC-HA/PTX3 vs. PBS control or nHC-HA/PTX3 vs. HA). (B) Cellviability. LPS-stimulated RAW264.7 cells were incubated on immobilizedPBS control, HA, or nHC-HA/PTX3 for 24 h (n=3). The cell viability wasmeasured by MTT assay. No significant differences (all p values > 0.05)in the cell viability among cells on these immobilized substrates wereobserved. (C) CD44 and TLR4 Receptors are responsible for attachment ofLPS-stimulated macrophages to immobilized nHC-HA/PTX3. RAW264.7 cells(2.5x10⁵/ml) were pre-incubated with the blocking antibody against CD44,TLR2, TLR4, integrin αv, β1, β2, or β3 or RGD peptides, along with theisotype control antibodies or a RGD control peptide, on ice for 30 min(n=3). After adding LPS (1 µg/ml), cells were incubated for 90 min andthe cell attachment assay was done the same as described in A. Anasterisk (∗) indicates p value < 0.05.

FIGS. 3A-E exemplifies polarization of LPS-stimulated macrophages towardM2 phenotype by immobilized nHC-HA/PTX3. (A) Relative mRNA expression ofM1 (TNF-α, IL-12p40) or M2 (IL-10, Arg-1, LIGHT, and SPHK1) markers inmacrophages bound to PBS control or immobilized HA or nHC-HA/PTX3 asdetermined by quantitative PCR. (B) Relative TNF-α protein amounts asdetermined by ELISA. (C) Western blot (left) and cytolocalization(right) by immunofluorescence staining of IRF5, which is a M1 marker, inmacrophages bound to PBS control or immobilized nHC-HA/PTX3. (D)Apoptosis of resting, fMLP- or LPS-stimulated neutrophils followingincubation with immobilized nHC-HA/PTX3. An asterisk (*) indicates p <0.05. (E) Phagocytosis of apoptotic neutrophils by resting orLPS-stimulated macrophages. An asterisk (*) indicates p < 0.05.

FIGS. 4A-B exemplifies the role of CD44 in maintaining M2 macrophagepolarization on immobilized nHC-HA/PTX3. (A) Relative mRNA expression ofM1 (IL-12p40) and M2 (IL-10, LIGHT, and SPHK1) macrophage markersfollowing binding of macrophages, pre-incubated with PBS or blockingantibodies to CD44, TLR4, or CD44/TLR4, to nHC-HA/PTX3 as determined byqPCR. An asterisk (*) indicates p < 0.05 compared to no antibodytreatment (none) in the same group. (B) IL-12 and IL-10 protein amountsas determined by ELISA. An asterisk (*) indicates p < 0.05 compared tono antibody treatment (none) in the same group.

FIGS. 5A-C exemplifies nHC-HA/PTX3(4^(th)) promotes the cellaggregation, but both nHC-HA/PTX3(2^(nd)) and nHC-HA/PTX3(4^(th))inhibit the production of IL-12p40 and IL-23 proteins inIFN-y/LPS-stimulated macrophages RAW264.7 cells (2.5x10⁵/ml) werecultivated on immobilized substrates (PBS as the control) and stimulatedwith IFN-γ/LPS for 4 h (A) or 24 h (B and C). (A) Cell morphology at 4 hafter seeding. Alternatively, cells are stimulated with LPS for 24 h andproteins of IL-10 and IL-12p70 in the cell culture supernatants weremeasured by respective ELISAs (B and C). p values are indicated in B andC.

FIGS. 6A-B exemplifies dose-dependent and covalent coupling of HMW HAand nHC-HA/PTX3 to surfaces of 96 well CovaLink™ plates. (A) HA ELISA ofbound HMW HA and nHC-HA/PTX3 after removal of unbound HMW HA andnHC-HA/PTX3. (B) HA ELISA of bound and unbound HA from HMW HA andnHC-HA/PTX3.

FIGS. 7A-B exemplifies dose-dependent binding of TSG-6 to immobilized HA(iHA) and resistance to various dissociating and reducing agents. (A)TSG-6 bound to iHA as measured by TSG-6 ELISA. (B) TSG-6 bound to iHA asmeasured by TSG-6 ELISA following treatment with 6M Guanidine HCl, 8MGuanidine HCl, 2% SDS, 100 mM DTT, or 25 mM NaOH.

FIGS. 8A-B exemplifies dose-dependent binding of PTX3 to immobilized HA(iHA) and resistance to various dissociating and reducing agents. (A)PTX3 bound to iHA as measured by PTX3 ELISA. (B) PTX3 bound to iHA asmeasured by PTX3 ELISA following treatment with 6M Guanidine HCl, 8MGuanidine HCl, 2% SDS, 100 mM DTT, or 25 mM NaOH.

FIG. 9 exemplifies the lack of competition or synergy between TSG-6 andPTX3 for binding iHA. The relative absorbance as measured by ELISA isshown for bound TSG-6 or PTX3 for incubation of each factor alone withiHA or combined incubation with iHA. No statistical significance isfound between alone and combined for either TSG-6 or PTX3 binding to iHA(p > 0.05).

FIGS. 10A-B exemplifies partial inhibition of PTX3 binding to iHApre-bound with TSG-6 and lack of inhibition of TSG-6 binding to iHApre-bound with PTX3. The figure shows TSG-6 and PTX3 ELISA results ofsubsequent binding of TSG-6 or PTX3 for pre-bound TSG-6/iHA (A) orpre-bound PTX3/iHA (B). P values are indicated in (A) and no statisticalsignificance was found among groups in (B).

FIG. 11 exemplifies attachment of LPS-stimulated RAW264.7 macrophages toPBS (control), HA (iHA), nHC-HA/PTX3, TSG-6/iHA or PTX3/iHA. Cells werephotographed 24 after incubation.

FIGS. 12A-E exemplifies relative gene expression in RAW264.7 macrophagesfollowing incubation on PBS (control), HA (iHA), nHC-HA/PTX3, TSG-6/iHAor PTX3/iHA. Total RNAs were isolated and mRNA expression of IL-12p40(A) and IL-10 (D) were measured by quantitative PCR. Alternatively,cells were stimulated with LPS (B and E) or IFN-γ/LPS (C) for 24 h, andprotein expression of IL-12p70 (B), IL-23 (C), and IL-10 (E) in cellculture media were measured using respective ELISAs. An asterisk (*)indicates p < 0.05 compared to the control.

FIGS. 13A-D exemplifies efficiency of TSG-6 free in solution versusbound TSG-6 for transferring HC1 and HC2 from IαI to iHA. (A, B)Relative bound HC1 (A) or IαI (B) following simultaneous or sequentialaddition of TSG-6 and IαI to iHA as determined by respective ELISA. Anasterisk (*) indicates p < 0.05 in the same TSG-6 concentration whenadded simultaneously and sequentially. (C) Western blot of samples fromA digested with hyaluronidase (HAase) and analyzed with anti-TSG-6antibody. (D) Relative HC1 and PTX3 bound to iHA following simultaneousincubation of PTX3 and IαI with iHA as determined by ELISA.

FIGS. 14A-G exemplifies complexes formed in solution followingsimultaneous incubation of IαI and TSG-6 with or without PTX3. (A-D)Western blot with antibodies against HC1 (A), HC2 (B), TSG-6 (C), orbikunin (D). HAase = treatment with hyaluronidase. (E) Illustration ofTSG-6 interaction with IαI. (F) Illustration of the inhibition ofHC2●TSG-6 formation by PTX3. (G) Western blot with antibody against IαI.

FIGS. 15A-G exemplifies complexes formed on iHA following simultaneousincubation of IαI and TSG-6 with or without PTX3. After washes with 8 MGnHCl and PBS, bound HC1, TSG-6, and PTX3 were measured by respectiveELISAs (A, D, F). An asterisk (*) indicates p < 0.05 compared to PTX3 at1 µg/ml. The complexes were washed again with 8 M GnHCl and PBS andbound components were digested with 1 unit/ml of hyaluronidase for 2 h.The digested samples were analyzed by Western blot with antibodiesagainst HC1 (B), HC2 (C), TSG-6 (E), and PTX3 (G).

FIGS. 16A-G exemplifies complexes formed on iHA following sequentialaddition of IαI with TSG-6 followed by PTX3. Bound HC1, TSG-6, and PTX3were measured by respective ELISAs (A, D, F). The complexes were washedagain with 8 M GnHCl and PBS and bound components were digested with 1unit/ml of hyaluronidase for 2 h. The digested samples were analyzed byWestern blot with antibodies against HC1 (B), HC2 (C), TSG-6 (E), PTX3(G).

FIGS. 17A-G exemplifies complexes formed on iHA following sequentialaddition of PTX3 followed by IαI with TSG-6. Bound HC1, TSG-6, and PTX3were measured by respective ELISAs (A, C, E). The complexes were washedagain with 8 M GnHCl and PBS and bound components were digested with 1unit/ml of hyaluronidase for 2 h. The digested samples were analyzed byWestern blot with antibodies against PTX3 (B), TSG-6 (D), HC1 (F), andHC2 (G).

FIG. 18 exemplifies attachment of LPS-stimulated RAW264.7 macrophages toPBS (control), HA (iHA), nHC-HA/PTX3, (IαI/TSG-6/PTX3)/iHA (IαI, TSG-6or PTX3 is simultaneously bound to iHA), (IαI/TSG-6)/PTX3/iHA(sequential addition of PTX3 to iHA pre-incubated with IαI and TSG-6),or (PTX3)/IαI/TSG-6/iHA (sequential addition of IαI and TSG-6 to iHApre-incubated with PTX3). Cells were photographed 24 after incubation.

FIGS. 19A-F exemplifies gene expression in RAW264.7 macrophagescultivated on immobilized substrates and stimulated with LPS. Total RNAswere isolated and expression of IL-10 and IL-12p40 mRNAs was measured byquantitative PCR (A and C). IL-10 and IL-12p70 proteins in the cellculture supernatants were measured by respective ELISAs (B and D). (E)IL-23 proteins in the cell culture supernatants of resting RAW264.7cells (none) or with stimulation of IFN-γ (200 units/ml), LPS (1 µg/ml),IFN-γ/LPS, LPS with immune complex (LPS/IC) or IL-4 (10 ng/ml) for 24 has measured by IL-23 ELISA. (F) IL-23 in the cell culture supernatantsof RAW264.7 cells cultivated on immobilized substrates and stimulatedwith IFN-γ/LPS for 24 h as measured by IL-23 ELISA. An asterisk (*)indicates p < 0.05.

FIGS. 20A-B exemplifies immunostaining of HA, PTX3, TSG-6, HCs andbikunin in human umbilical cord (A) or amniotic membrane (B). Frozensections of human umbilical cord were probed with biotinylated HABP withor without HAase digestion and with antibodies against PTX3 and TSG-6,and chain-specific antibodies against IαI and PαI components asindicated. Nuclei were counter-stained with Hoechst 33342 (blue). Epi,Epithelium. Bar represents 100 µm.

FIGS. 21A-B exemplifies a comparison of PTX3 levels in sequential PBSand GnHCl extract from AM, CH and UC. A, each lane contains 2 µg HA inlanes 2 and 3 and 20 µg total proteins in lanes 4-11. B, each lanecontains 40 µg total proteins in lanes 3-10.

FIGS. 22A-C exemplifies a comparison of HC1, bikunin and IαI insequential PBS and GnHCl extracts from AM, CH and UC. Each lane contains20 µg total protein in A and C and 40 µg total protein in B and D-Fexcept positive control.

FIG. 23 exemplifies a comparison of TSG-6 in AM and UC GnHCl extract.Each lane contains 40 µg total proteins except positive TSG-6 control.

FIGS. 24A-D exemplifies Western blot analysis of PTX3 (A), HC1 (B), HC2(C), and TSG-6 (D) in 1-4th AM HC●HA complex. Each lane contains 4 µg HAexcept positive control.

FIG. 25 exemplifies Western blot analysis of TSP-1 in AME, AM GnHCl and1-4th HC-HA complex. Lanes 3, 4, 10 and 11, each lane contains 30 µgtotal proteins. Lanes 5-8 and 12-15, each lane contains 4 µg HA.

FIGS. 26A-E exemplifies Western blot analysis of PTX3 (A), HC1 (B), HC2(C), HC3 (D) and TSG-6 (E) in 4th UC HC-HA complex. Each lane contains 4µg HA except positive control.

FIGS. 27A-B exemplifies a comparison of HC1 (A) and PTX3 (B) in 4thHC●HA complex from PBS and GnHCl extract. Each lane contains 4 µg HAexcept positive control.

FIG. 28 exemplifies a comparison of 4th HC-HA complex from PBS and GnHClin agarose gel. Each lane contains 15 µg HA except positive HA control.

FIGS. 29A-B exemplifies Coomassie blue staining for SDS-PAGE gel ofGnHCl HC-HA and PBS HC-HA. A. AM PBS and GnHCl HC-HA. B. UC PBS andGnHCl HC-HA. Each lane contains 30 µg HA.

FIGS. 30A-D exemplifies that keratan sulfate and osteoadherin werepresent in AM GnHCl HC-HA but not in PBS HC-HA. A. Coomassie bluestaining. Each lane contains 30 µg HA. B and C. Western blot for keratansulfate (B) and osteoadherin (C). Each lane contains 4 µg HA. D,Immunostaining for keratin sulfate in AM.

FIGS. 31A-H exemplifies deglycosylation and analysis of AM GnHCl HC-HAby SDS-PAGE with (Coomassie Blue) CB staining or Western blots. A.Coomassie blue staining. Each lane contains 30 µg HA except lane 6 whichcontains 5 µg HA. B-H. Western blots for Osteoadherin (B), Decorin (C,D), Biglycan (E, F), Keratan sulfate (G) and PTX3 (H). H: hyaluronidase;C: chondroitinase (Cabc); K: keratinase (keratan sulfateendo-β-galactosidase); T: TFMSA (trifluoromethanesulfonic acid). Eachlane contains 4 µg HA.

FIGS. 32A-G exemplifies Decorin and biglycan were abundantly present inUC GnHCl HC-HA but not in PBS HC-HA. Keratan sulfate, osteoadherin andbikunin also were present in UC GnHCl HC-HA but not in PBS HC-HA exceptfor keratan sulfate. A. Coomassie blue staining. Each lane contains 30µg HA except lane 6 which contains 5 µg HA. B-H. Western blots forDecorin (B), Biglycan (C), Bikunin (D), PTX3 (E), Keratan sulfate (F)and Osteoadherin (G). H: hyaluronidase; C: chondroitinase (Cabc); K:keratinase (keratan sulfate endo-β-galactosidase). Each lane contains 4µg HA.

FIG. 33 exemplifies immunolocalization of PTX3 in human AM. Frozensections of human fetal membrane were probed with anti-PTX3,biotinylated HABP with or without HAase digestion and withchain-specific antibodies against IαI components. Nuclei werecounter-stained with Hoechst 33342 (blue). AM, amniotic membrane; Epi,Epithelium; CH, chorion. Bar, represents 100 µm.

FIGS. 34A-C exemplifies presence of PTX3 in AM soluble extract andpurified HC-HA complex. Purified PTX3, AM extract (AME) and AM HC-HAcomplex were treated with or without 50 mM NaOH at 25° C. for 1 h orhyaluronidase (HAase) at 37° C. for 1 h before Western blotting usinganti-PTX3 (A) and anti-HC1 (B) antibodies and analysis on 0.5% agarosegel electrophoresis before staining with Stains-all dye (C) . PTX3species and its multimeric form were found in AM soluble extract andpurified HC-HA complex. M, protein ladder markers.

FIGS. 35A-C exemplifies expression of PTX3 mRNA and protein by AMECs andAMSCs. RNA and protein were extracted from human skin fibroblasts (SkinFib.), and both AMECs and AMSCs. Expression of PTX3 mRNA (A) and proteinin supernatants and cell lysates (B) was compared. PTX3 siRNAtransfection was performed to verify the expression of PTX3 in AMECs andAMSCs (C).

FIG. 36 exemplifies morphological changes of human skin fibroblasts(HSF, A), AMSC (B) and AMEC (C) after agarose overlay. HSF, AMSC andAMEC were cultured in both serum-free and serum-containing conditionswith or without a 3% agarose overlay for five days and the cellmorphology were photographed. Scale bar, 50 µm.

FIG. 37 exemplifies that agarose overlay decreased the release of HAinto culture media by HSF, AMSC and AMEC cultures. The HA concentrationwere measured by ELISA assay in culture media from HSF, AMSC and AMECwith or without agarose overlay in both serum-free and serum-containingconditions.

FIG. 38 exemplifies immunolocalization of PTX3, HA and HC1 in cellcultures with an agarose overlay. HSF, AMSC and AMEC were cultured withan agarose overlay with or without TNF treatment and probed forhyaluronan (red), PTX3 (green, A-F; red, J-L) and HC1 (green) (nucleiare blue). Colocalization of HC1 with HA were found in all cultures, butcolocalization of PTX3 with HA or HC1 were only found in AMSC and AMEC.Scale bar, 50 µm.

FIG. 39 exemplifies HC-HA/PTX3 complex in AMSC but not HSF under agaroseoverlay. GnHCl extracts of cell layers from agarose overlayed HSF andAMSC cultures were subjected to Western blot for PTX3 with or with NaOHtreatment. M, protein ladder markers.

FIGS. 40A-C exemplifies reconstitution of HC-HA/PTX3 complex onimmobilized HA in vitro. iHA (~ 14 µg/ml), IαI (5 µg/ml), and TSG-6 (12µg/ml) were incubated simultaneously without or with PTX3 (1, 5, or 20µg/ml) for 2 h at 37° C. For sequentially, iHA (~ 14 µg/ml), IαI (12µg/ml), and TSG-6 (12 µg/ml) were incubated in the reaction buffer for 2h at 37° C., then PTX3 (1, 5, or 20 µg/ml) were added and incubated foranother 2 h at 37° C. After washes with 8 M GnHCl and PBS, iHA withbound components were digested with 1 unit/ml of hyaluronidase for 2 hat 60° C. The samples were analyzed by Western blot with antibodiesagainst IαI (A), TSG-6 (B) and PTX3 (C).

FIGS. 41A-B exemplifies cell morphology of human corneal fibroblasts upto D3 cultured on DMEM/10% FBS for 2 Days (A) or DMEM/10% FBS for 2 Days± TGF-β1 for 3 Days (B).

FIG. 42 exemplifies soluble HC-HA (PBS) inhibits TGFβ1 but activatesTGFβ3 signaling while insoluble HC-HA (GnHCl) activates both TGFβ1 andTGFβ3 signaling under serum-free conditions and is further enhanced withTGFβ1 stimulation.

FIG. 43 exemplifies both soluble HC-HA (PBS) and insoluble HC-HA (GnHCl)inhibit TGFβR2 and TGFβR3 expression under challenge of TGFβ1. A, TGFβRmRNA expression. B, TGFβR protein expression.

FIG. 44 exemplifies inhibition of nuclear translocation of pSMAD⅔signaling by HC-HA inhibition of TGFβ1 signaling.

FIG. 45 exemplifies HC-HA inhibition of alpha smooth muscle actinformation.

FIG. 46 exemplifies BMP6 transcript was activated by HA andsoluble/insoluble HC-HA. Addition of TGFβ1 activates transcriptexpression of BMP6 on plastic but dramatically activates mRNA expressionof BMP4/6 in HCF on HA and both soluble and insoluble HC-HA.

FIG. 47 exemplifies HC-HA but not HA activates transcript expression ofBMPR1A in HCF challenged with TGFβ1, while additionalTGFβ1non-specifically activates mRNA expression of BMPR1B and BMPR2 inHCF.

FIG. 48 exemplifies both soluble HC-HA (PBS) and insoluble HC-HA (GnHCl)activates BMP4/6 signaling via pSMAD1/5/8.

FIG. 49 exemplifies activation of SMAD/1/5/8 resulted in upregulation ofits downstream gene, inhibitor of DNA binding 1, 3 and 4 (ID1, ID3 andID4), downstream targets of BMP signaling.

FIG. 50 exemplifies HC-HA (PBS) and HC-HA (GnHCl) promote Keratocan mRNAexpression by 14- and 16-fold respectively, which was significantlydownregulated by additional TGFβ1.

FIG. 51 exemplifies HC-HA (PBS) and HC-HA (GnHCl) promote keratocanprotein expression.

FIG. 52 exemplifies HCF express more ESC markers on 4X HC-HA (PBS andinsoluble HC-HA (GnHCl) than on plastic and when the cells werechallenged with addition of TGFβ1.

FIG. 53 exemplifies cell viability of MC3T3-E1 cells as measured by MTT.

FIG. 54 exemplifies mineralization of MC3T3-E1 cells as measured byAlizarin Red Staining.

FIGS. 55A-B exemplifies morphology of MC3T3-E1 cells treated with HC-HA(A) or AMP (B) on Day 13 of induction.

FIGS. 56A-B exemplifies Alizarin Red staining of MC3T3-E1 cells treatedwith HC-HA (A) or AMP (B) on day 13 of induction.

FIGS. 57A-B exemplifies quantitative analysis of ARS staining ofMC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction.

FIGS. 58A-B exemplifies ALP activity (IU/L) of HC-HA (A) and AMP (B)treated cells on day 13 of induction.

FIGS. 59A-B exemplifies phase contrast microscopy of morphologicalchanges in MC3T3-E1 cells following induction from Day 3. (A) Uninducedcells were cultured in αMEM w/ 10% FBS for 7 days. (B) MC3T3-E1 cellswere cultured to confluence in flat bottom 96-well plates one day afterseeding (Day 0). Cells were then induced with ascorbic acid andβ-glycerophosphate.

FIGS. 60A-B exemplifies phase contrast microscopy of morphologicalchanges in induced MC3T3-E1 cells treated with HC-HA (A) or AMP (B) fromDay 1 to Day 7.

FIGS. 61A-B exemplifies phase contrast microscopy of spindle ringformation in induced MC3T3-E1 cells from Day 3 of induction.

FIGS. 62A-B exemplifies phase contrast microscopy of spindle ringformation in induced MC3T3-E1 cells treated with HC-HA (A) and AMP (B)(Day 0 to Day 6).

FIG. 63 exemplifies ARS staining of induced MC3T3-E1 cells treated withHC-HA and AMP and Extraction with GnHCl.

FIGS. 64A-B exemplifies ARS Extraction and Quantitation of MC3T3-E1cells with Acetic Acid and 10% Ammonium Hydroxide. ARS extracts throughacetic acid treatment were neutralized with 10% ammonium hydroxide thenadded to 96-well clear bottom assay plates (A) for reading on aspectrophotometer (B). * denotes statistical significance.

FIGS. 65A-B exemplifies ARS Extraction and Quantitation of MC3T3-E1cells with GnHCl. ARS extracts through GnHCl treatment were added to96-well clear bottom assay plates (A) for reading on a spectrophotometer(B). * denotes statistical significance.

FIGS. 66A-B exemplifies ARS staining (A) and quantitation (B) ofMC3T3-E1 cells treated with HC-HA on D19 (D18 of Induction). ARSstaining was conducted on D19 of culturing (D18 Induction).

FIGS. 67A-B exemplifies ARS staining (A) and quantitation (B) ofMC3T3-E1 cells treated with AMP. ARS staining was conducted on D19 ofculturing (D18 Induction).

FIG. 68 exemplifies a spindle like ring observed in induced MC3T3-E1cells on Day 14 of induction.

FIGS. 69A-B exemplifies phase contrast microscopy of cell morphology ofinduced MC3T3-E1 cells treated with AMP after 14 days of inductioncultured (A) without a transwell or (B) with a transwell.

FIG. 70 exemplifies ARS Staining of induced MC3T3-E1 cells at D14 ofinduction.

FIG. 71 exemplifies quantitation of ARS Staining of induced MC3T3-E1cells at D14 of induction.

FIGS. 72A-B exemplifies ARS staining and quantitation of MC3T3-E1 cellstreated with AMP (A) Phase contrast picture and ARS staining pictureMC3T3-E1 cells taken on Day 21 culturing (Day 20 of induction). (B) ARSstaining was quantified on Day 21 of culture (Day 20 of induction).The * symbol denotes statistical significance of p < 0.05.

FIG. 73 illustrates a map of different progenitor differentiation toosteoblast, with common factors added to culture medium shown.

FIGS. 74A-B exemplifies ARS staining and quantitation in MC3T3-E1 cells.(A) Phase contrast micrographs with or without ARS staining of HUVEC,hBMMSCs, and hAM stromal stem cells from Day 4 to Day 21. (B)Quantitation of ARS staining. The * symbol denotes statisticalsignificance of p < 0.05 when compared to the negative control.

FIG. 75 illustrates a timeline of osteogenesis in MC3T3-E1 cells.

FIGS. 76A-B exemplifies ARS staining and quantitation in MC3T3-E1 cells.(A) Cell morphology and ARS staining of MC3T3-E1 cells treated with AMP(Day 1, 2, 7, 10). (B) ARS Quantitation of MC3T3-E1 cells treated withAMP (Day 1, 2, 7, 10) The * symbol denotes statistical significance of p< 0.05.

FIGS. 77A-B exemplifies a timeline of cell viability and proliferationthrough MTT assay. (A) MTT Assay of MC3T3-E1 cell viability andmetabolic activity on Day 1, 2, and 4. The * symbol denotes statisticalsignificance of p < 0.05 from Day 1. (B) BrdU Assay of MC3T3-E1 cellproliferation on Day 1, 2, and 16. The * symbol denotes statisticalsignificance of p < 0.05 from Day 1.

FIGS. 78A-B exemplifies mRNA expression by QPCR for various genes testedfollowing differentiation induction with or without AMP treatment. (A)hMSC and (B) MC3T3-E1 cell expression.

FIGS. 79A-C exemplifies phase microscopy and quantitation of ARS Assay.MC3T3-E1 cells deposited mineralization for the duration of theexperiment, 8 days. ARS was then used for qualitative analysis. (A)CovaLink-NH 96-well plate in which the PBS, HA, and HC-HA wereimmobilized. (B) Conventional 96-well plate with the negative controland AGM (inductive agents) and AMP as positive controls. (C)Quantitation of ARS assay. The * symbol denotes statistical significanceof p < 0.05 from Day 1.

FIGS. 80A-E exemplifies mRNA expression by QPCR for various genes testedfollowing differentiation induction with or without HC-HA/PTX3treatment.(A) Master transcription factors Runx2 and Sox9 forosteogenesis and chondrogenesis, Day2 1, 7, and 14. (B) Bone morphogenicproteins (BMPs) expression on Day 14. (C) Chondrogenic marker Collagen 2(COL2) and osteogenic marker alkaline phosphatase (ALPL), expressed onDays 7 and 14. (D) Hypertrophic markers Collagen 10 (COL10) and MMP13expressed on Day 14. (E) Osteogenic markers Collagen 1 (COL1), Osterix(OSX) and Bone Sialoprotein (BSP) on Day 14.

FIGS. 81A-B exemplifies phase microscopy (A) and quantitation (B) of ARSAssay following differentiation induction with or without HC-HA/PTX3(soluble or insoluble) treatment on Day 14.

FIG. 82 exemplifies CD4⁺ T cell activation and differentiation. Underdifferent stimuli, Naïve CD4⁺ T helper cell (Th) is differentiated intoTh1, Th2, Th17, or Treg and secreted different cytokines. Th1-typecytokines (e.g., IFN-γ and IL-2) tend to produce the pro-inflammatoryresponses.

FIG. 83 exemplifies the procedure to measure cell proliferation andcytokine production. Splenocytes were isolated from OT-II mice thatexpress a transgenic TCR specific for ovalbumin (OVA), and stimulatedwith OVA up to 4 days. Cell proliferation was measured by BrdU labelingand expression of cytokines (IFN-γ and IL-2) was measured by therespective ELISA.

FIG. 84 exemplifies nHC-HA/PTX3 inhibition of CD4⁺ T cell proliferation.Splenocytes isolated from Ova T cell receptor transgenic mice werestimulated with OVA (0 -10 µM) for 4 days. AM extract (AME, 25 µg/ml)and nHC-HA/PTX3 (25 µg/ml) inhibited the clone growth of activated Tcells induced by increasing OVA concentrations (top). Proliferation ofsplenocytes treated with HA, AME, or nHC-HA/PTX3 and labeled with CFSEfor 4 days was measured by flow cytometry. Both AME and nHC-HA/PTX3dose-dependently inhibited the cell proliferation (middle). 25 µg/mlnHC-HA/PTX3 inhibited the proliferation of CD4⁺ T cell labeled with BrdU(bottom) (∗, p < 0.05 compared to the control).

FIG. 85 exemplifies nHC-HA/PTX3 suppression of Th1-type cytokines IFN-γand IL-2. Splenocytes treated with PBS, 25 µg/ml HA, 25 µg/ml AME, or 25µg/ml nHC-HA/PTX3 were stimulated with 10 µM OVA for 4 days. IFN-γ andIL-2 in culture supernatants were measured by the respective ELISA. BothAME and nHC-HA/PTX3 suppressed the production of IFN-γ and IL-2 (∗ p <0.05 compared to the control).

FIG. 86 exemplifies nHC-HA/PTX3 reduction of macrophage (labeled withenhanced green fluorescent protein, or EGFP) influx. LPS (2 µl of 2µg/ml) was injected into the corneal stroma of Mafia mouse. Immediately,5 µl of PBS or nHC-HA/PTX3 (1 mg/ml) was injected into each quadrant ofone cornea from the same mouse through the subjunctival tissue. Theinflux of EGFP⁺ macrophages is monitored using in vivo intravitalmicroscopy at day 1, 2, 3, and 6 after LPS treatment (top).Alternatively, mouse corneas were treated with PBS or nHC-HA/PTX3simultaneously with LPS (pretreatment (-) or three days before LPStreatment (pretreatment (+)). At day 4 after LPS treatment, cells incorneas were isolated by collagenase digestion and sorted into EGFP⁻ orEGFP⁺ (macrophages) by FACS (* and **, p < 0.05 and p < 0.01 compared tothe control, respectively).

FIG. 87 exemplifies nHC-HA/PTX3 polarization of macrophages toward a M2phenotype. mRNA expression of M2 markers (Arg-1 and IL-10) and M1markers (IL-12p40 and IL-12p35) in macrophages (EGFP⁺) infiltrated toLPS-elicited murine corneas was quantitated by qPCR (∗ and **, p < 0.05and p < 0.01 compared to the control, respectively).

FIG. 88 exemplifies nHC-HA/PTX3 improvement of corneal allograftsurvival. The survival of murine corneal allograft was significantlyimproved by injection of nHC-HA/PTX3 at one quadrant subconjunctivalsite (10 µg /time, two times /a week, top left), but even dramaticallybetter by injection of nHC-HA/PTX3 at four quadrant subconjunctivalsites (20 µg /time, two times / a week, top right). Bottom, photographsof PBS (post operative day 21, left) or nHC-HA/PTX3 treatment (postoperative day 40, right) in corneal allografts.

FIG. 89 exemplifies macrophage classical M1 activation (e.g., induced byIFN-γ and/or TLR ligands such as LPS) to express high levels ofproinflammatory cytokines (such as TNF-α, IL-12, and IL23), whichactivate Th1 and Th17 lymphocytes leading to many chronic inflammatorydiseases.

FIGS. 90A-D exemplifies LPS-elicited macrophage infiltration to murinecorneas with treatment of PBS (control), HC-HA/PTX3, or AMP. Mafia mice(macrophages are EGFP+) with LPS intrastromal injection (5 µg) for botheye. OS was treated with PBS, OD was treated with either HC-HA (2injection sites) (A), HC-HA (4 injection sites) (B), AMP (2 injectionsites) (C), AMP (4 injection sites) (D), each injection was 5 µl.Treatment was one time right after LPS injection. Images were taken onday 1, day 2, day 3 and day 6. Cells were counted based on the intensityof green fluorescence.

FIGS. 91A-B exemplifies LPS-elicited macrophage infiltration to murinecorneas and their subtypes (M1 and M2) with treatment or pretreatment ofPBS (control), nHC-HA/PTX3, or AMP. Pretreatment and treatment of Mafiamice are the same as described in FIG. 86 . On Day 4, cornea buttons arecut and digested with collagenase at 37° C. for 2 h.EGFP-positive/negative cells are sorted out by FACS. The ratio ofEGFP-positive macrophages to EGFP-negative cells is calculated and usedas an arbitrary unit to determine the extent of macrophage infiltration(A). Total RNA was extracted from sorted EGFP-positive macrophages andconverted to cDNAs. The expression of Arg-1, IL-10, IL-12p40, andIL-12p35 is measured by quantitative PCR (B).

FIG. 92 Diagram of injection site. The injection locations will besubconjunctiva close to fornix nHC-HA/PTX3 or AMP can reduce DS-inducedALKC in murine experimental dry eye model.

FIG. 93 exemplifies HC-HA activation of IGF1-HIF1α-VEGF signaling topromote angiogenesis. HC-HA induces 2- to 6-fold increase of IGF1 mRNAand 2-fold increase of VEGF mRNA when the cells are in rest condition.HC-HA induces 5- to 12-fold increase of IGF1 mRNA and 5- to 9-foldincrease of VEGF mRNA when the cells are challenged by TGFβ (10 ng/ml).n=4, ^(∗)p<0.05, **p<0.01. IGF1, Insulin-like growth factor 1; HIF1α,Hypoxia-inducible factor 1-alpha; VEGF, Vascular endothelial growthfactor.

DETAILED DESCRIPTION OF THE INVENTION Certain Terminology

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the claimed subject matter belongs. All patents, patentapplications, published applications and publications, GENBANKsequences, websites and other published materials referred to throughoutthe entire disclosure herein, unless noted otherwise, are incorporatedby reference in their entirety. In the event that there is a pluralityof definitions for terms herein, those in this section prevail. Wherereference is made to a URL or other such identifier or address, it isunderstood that such identifiers can change and particular informationon the internet can come and go, but equivalent information is known andcan be readily accessed, such as by searching the internet and/orappropriate databases. Reference thereto evidences the availability andpublic dissemination of such information.

As used herein, ranges and amounts can be expressed as “about” aparticular value or range. About also includes the exact amount. Hence“about 5 µg” means “about 5 µg” and also “5 µg.” Generally, the term“about” includes an amount that would be expected to be withinexperimental error.

As used herein, a reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) complex is anHC-HA/PTX3 complex that is formed by assembly of the component moleculesof the complex in vitro. The process of assembling the rcHC-HA/PTX3includes reconstitution with purified native proteins or molecules frombiological source, recombinant proteins generated by recombinantmethods, or synthesis of molecules by in vitro synthesis. In someinstances, the purified native proteins used for assembly of thercHC-HA/PTX3 are proteins in a complex with other proteins (i.e. amultimer, a multichain protein or other complex). In some instances,PTX3 is purified as a multimer (e.g. a homomultimer) from a cell andemployed for assembly of the rcHC-HA/PTX3 complex.

As used herein, a purified native HC-HA/PTX3 (nHC-HA/PTX3) complexrefers to an HC-HA/PTX3 complex that is purified from a biologicalsource such as a cell, a tissue or a biological fluid. Such complexesare generally assembled in vivo in a subject or ex vivo in cells,tissues, or biological fluids from a subject, including a human or otheranimal.

As used herein, a PTX3/HA complex refers to an intermediate complex thatis formed by contacting PTX3 with immobilized HA. In the methodsprovided herein, the PTX3/HA complex is the generated prior to theaddition of HC1 to HA.

As used herein, “hyaluronan,” “hyaluronic acid,” or “hyaluronate” (HA)are used interchangeably to refer to a substantially non-sulfated linearglycosaminoglycan (GAG) with repeating disaccharide units ofD-glucuronic acid and N-acetylglucosamine(D-glucuronosyl-N-acetylglucosamine).

As used herein, the term “high molecular weight” or “HMW,” as in highmolecular weight hyaluronan (HMW HA), is meant to refer to HA that has aweight average molecular weight that is greater than about 500kilodaltons (kDa), such as, for example, between about 500 kDa and about10,000 kDa, between about 800 kDa and about 8,500 kDa, between about1100 kDa and about 5,000 kDa, or between about 1400 kDa and about 3,500kDa. In some embodiments, the HMW HA has a weight average molecularweight of 3000 kDa or greater. In some embodiments, the HMW HA has aweight average molecular weight of 3000 kDa. In some embodiments, theHMW HA is Healon® with a weight average molecular weight of about 3000kDa. In some embodiments, HMW HA has a molecular weight of between about500 kDa and about 10,000 kDa. In some embodiments, HMW HA has amolecular weight of between about 800 kDa and about 8,500 kDa. In someembodiments, HMW HA has a molecular weight of about 3,000 kDa.

As used herein, the term “low molecular weight” or “LMW,” as in lowmolecular weight hyaluronan (LMW HA), is meant to refer to HA that has aweight average molecular weight that is less than 500 kDa, such as forexample, less than about 400 kDa, less than about 300 kDa, less thanabout 200 kDa, about 200-300 kDa, or about 1-300 kDa.

As used herein, pentraxin 3, or PTX3, protein or polypeptide refers toany PTX3 protein, including but not limited to, a recombinantly producedprotein, a synthetically produced protein, a native PTX3 protein, and aPTX3 protein extracted from cells or tissues. PTX3 include multimericforms (e.g. homomultimer) of PTX3, including, but not limited to,dimeric, trimeric, tetrameric, pentameric, hexameric, tetrameric,octameric, and other multimeric forms naturally or artificiallyproduced.

As used herein, Tumor necrosis factor Stimulated Gene-6 (TSG-6) refersto any TSG-6 protein or polypeptide, including but not limited to, arecombinantly produced protein, a synthetically produced protein, anative TSG-6 protein, and a TSG-6 protein extracted from cells ortissues.

As used herein, inter-α-inhibitor (IαI) refers to the IαI proteincomprised of light chain (i.e., bikunin) and one or both heavy chains oftype HC1 or HC2 covalently connected by a chondroitin sulfate chain. Insome embodiments, the source of IαI is from serum or from cellsproducing IαI e.g., hepatic cells or amniotic epithelial or stromalcells or umbilical epithelial or stromal cells under a constitutive modestimulation by proinflammatory cytokines such as IL-1 or TNF-α.

As used herein, a “hyaluronan binding protein”, “HA binding protein”, or“HABP” refers to any protein that specifically binds to HA.

As used herein, “link module” means a hyaluronan-binding domains.

As used herein, “biological activity” refers to the in vivo activitiesof an nHC-HA/PTX3 or rcHC-HA/PTX3 complex or physiological responsesthat result upon in vivo administration of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex or a composition or mixture containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex. Biological activity, thus,encompasses therapeutic effects and pharmaceutical activity ofnHC-HA/PTX3 or rcHC-HA/PTX3 complexes and compositions and mixturesthereof.

As used herein, the terms “subject”, “individual” and “patient” are usedinterchangeably. None of the terms are to be interpreted as requiringthe supervision of a medical professional (e.g., a doctor, nurse,physician’s assistant, orderly, hospice worker). As used herein, thesubject is any animal, including mammals (e.g., a human or non-humananimal) and non-mammals. In one embodiment of the methods andcompositions provided herein, the mammal is a human.

As used herein, the terms “treat,” “treating” or “treatment,” and othergrammatical equivalents, include alleviating, abating or amelioratingone or more symptoms of a disease or condition, ameliorating, preventingor reducing the appearance, severity or frequency of one or moreadditional symptoms of a disease or condition, ameliorating orpreventing the underlying metabolic causes of one or more symptoms of adisease or condition, inhibiting the disease or condition, such as, forexample, arresting the development of the disease or condition,relieving the disease or condition, causing regression of the disease orcondition, relieving a condition caused by the disease or condition, orinhibiting the symptoms of the disease or condition eitherprophylactically and/or therapeutically. In a non-limiting example, forprophylactic benefit, an rcHC-HA/PTX3 complex or composition disclosedherein is administered to an individual at risk of developing aparticular disorder, predisposed to developing a particular disorder, orto an individual reporting one or more of the physiological symptoms ofa disorder.

As used herein, “placenta” refers to the organ that connects adeveloping fetus to the maternal uterine wall to allow nutrient uptake,waste elimination, and gas exchange via the maternal blood supply. Theplacenta is composed of three layers. The innermost placental layersurrounding the fetus is called amnion. The allantois is the middlelayer of the placenta (derived from the embryonic hindgut); bloodvessels originating from the umbilicus traverse this membrane. Theoutermost layer of the placenta, the chorion, comes into contact withthe endometrium. The chorion and allantois fuse to form thechorioallantoic membrane.

As used herein, “chorion” refers to the membrane formed byextraembryonic mesoderm and the two layers of trophoblast. The chorionconsists of two layers: an outer formed by the trophoblast, and an innerformed by the somatic mesoderm; the amnion is in contact with thelatter. The trophoblast is made up of an internal layer of cubical orprismatic cells, the cytotrophoblast or layer of Langhans, and anexternal layer of richly nucleated protoplasm devoid of cell boundaries,the syncytiotrophoblast. The avascular amnion is adherent to the innerlayer of the chorion.

As used herein, “amnion-chorion” refers to a product comprising amnionand chorion. In some embodiments, the amnion and the chorion are notseparated (i.e., the amnion is naturally adherent to the inner layer ofthe chorion). In some embodiments, the amnion is initially separatedfrom the chorion and later combined with the chorion during processing.

As used herein, “umbilical cord” refers to the organ that connects adeveloping fetus to the placenta. The umbilical cord is composed ofWharton’s jelly, a gelatinous substance made largely frommucopolysaccharides. It contains one vein, which carries oxygenated,nutrient-rich blood to the fetus, and two arteries that carrydeoxygenated, nutrient-depleted blood away.

As used herein, “placental amniotic membrane” (PAM) refers to amnioticmembrane derived from the placenta. In some embodiments, the PAM issubstantially isolated.

As used herein, “umbilical cord amniotic membrane” (UCAM) means amnioticmembrane derived from the umbilical cord. UCAM is a translucentmembrane. The UCAM has multiple layers an epithelial layer, a basementmembrane; a compact layer; a fibroblast layer; and a spongy layer. Itlacks blood vessels or a direct blood supply. In some embodiments, theUCAM comprises Wharton’s Jelly. In some embodiments, the UCAM comprisesblood vessels and/or arteries. In some embodiments, the UCAM comprisesWharton’s Jelly and blood vessels and/or arteries.

As used herein, the terms “purified”, “and “isolated” mean a material(e.g., nHC-HA/PTX3 complex) substantially or essentially free fromcomponents that normally accompany it in its native state. In someembodiments, “purified” or “isolated” mean a material (e.g., nHC-HA/PTX3complex) is about 50% or more free from components that normallyaccompany it in its native state, for example, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% free from from components thatnormally accompany it in its native state.

Overview: nHC-HA/PTX3 and rcHC-HA/PTX3 Complexes

Hyaluronan (HA) is a substantially non-sulfated linear glycosaminoglycan(GAG), composed of repeating disaccharide subunits units of D-glucuronicacid and N-acetyl-D-glucosamine via GlcUA-β1,3-GlcNAc- β1,4-linkages. HAis synthesized by HA synthases (e.g., HAS1, HAS2, and HAS3) anddeposited into the extracellular matrix, where it contributes to thestructural integrity of tissues and also regulates many cellular processvia interaction with proteins, including cell surface receptors. Themolecular weight of HA typically ranges in size from about 200 to about10,000 kDa. Normal levels of HA are maintained in tissues through abalance of biosynthesis by HAS enzymes and catabolism by hyaluronidases,such as Hyal1.

High molecular weight HA (HMW HA), typically greater than 500 kDa,promotes cell quiescence and structural integrity of such tissues as thecartilage and the vitreous body (humor) in the eye, and is associatedwith scarless fetal wound healing. In certain instances, HMW HA inhibitsthe gene expression of pro-inflammatory mediators and angiogenesis.

In certain pathogenic conditions, HMW HA is degraded into smallerfragments and oligosaccharides (e.g., via hyaluronidase or free radicaloxidation). LMW HA fragments stimulate vascular endothelial cellproliferation, migration, collagen synthesis, sprout formation, andangiogenesis in rat skin, myocardial infarction, and cryo-injured skingraft model by promoting the gene expression of pro-inflammatory andpro-angiogenic mediators.

The biological functions of HA are mediated though interaction of HAwith HA-binding proteins (HABPs), also called hyaladherins. Suchproteins include, but are not limited to, tumor necrosisfactor-α-stimulated gene 6 (TSG-6), aggrecan, versican, neurocan,brevican, LYVE-1, CD44, and inter-α- inhibitor (IαI). In some instances,HABPs comprise a link module domain that binds to HA. TSG-6, aggrecan,versican, neurocan, brevican, LYVE-1 and CD44 are exemplary HABPs thatcontain a link module.

IαI comprises two heavy chains (HC1 and HC2), both of which are linkedthrough ester bonds to a chondroitin sulfate chain that is attached to alight chain (i.e., Bikunin). In some instances, HA forms a covalentcomplex (hereinafter, “HC-HA”) with one or both of the HCs of IαI bycovalent linkage to the IαI heavy chains. In certain instances, the IαIis found in serum and/or obtained from cells producing IαI, e.g.,hepatic cells or amniotic epithelial or stromal cells or umbilicalepithelial or stromal cells under a constitutive mode stimulation byproinflammatory cytokines, such as IL-1 or TNF-α

In certain instances, TSG-6 facilitates the transfer of, catalyzes thetransfer of, and/or transfers the HC1 and HC2 of IαI to HA. TSG-6 formsstable complexes with immobilized HA (TSG-6●HA) resulting in thetransfer HC1 and HC2 to HA to form an HC-HA complex and release of TSG-6from the complex. The expression of TSG-6 is often induced byinflammatory mediators such as TNF-α and interleukin-1 and duringinflammatory-like processes such as ovulation and cervical ripening.

Amniotic membrane (AM) modulates adult wound healing and facilitatestissue regeneration. In certain instances, AM promotes epithelializationwhile suppressing stromal inflammation, angiogenesis and scarring. AMhas been used successfully as a surgical graft or temporary biologicalpatch for the treatment of ophthalmic conditions which require cornealand conjunctival surface reconstruction, including, but not limited to,persistent epithelial defect, deep corneal ulcer, infectious keratitis,symptomatic bullous keratopathy, acute Stevens Johnson Syndrome/ToxicEpidermal Necrolysis (SJS/TEN), limbal stem cell deficiency, pterygium,pinguecula, conjunctivochalasis, symblepharon, formix reconstruction,and conjunctival tumors.

The avascular stromal matrix of AM contains high amounts of HA andconstitutively expresses IαI (Zhang et al. (2012) J. Biol. Chem.287(15):12433-44). HMW HA in AM forms nHC-HA complexes (He et al. (2009)J. Biol. Chem. 284(30):20136-20146). As shown herein in the Examplesprovided, this nHC-HA complex also contains pentraxin 3, PTX3 (FIG. 1 ),and hence it is referred to herein as “nHC-HA/PTX3 complex.” NativeHC-HA/PTX3 complexes extracted from the AM exhibit suppression of TGF-βpromoter activity, promotion of macrophage cell death, and suppressionof blood vessel development. The nHC-HA/PTX3 complexes of the AM thusserve an active role in AM’s anti-inflammatory, antiscarring andantiangiogenic actions.

As described herein, nHC-HA/PTX3 complexes are also found in theumbilical cord (UC). The UC HC-HA/PTX3 complexes differ in theirbiochemical composition with respect to HA content and the presenceand/or relative abundance of various components of the complex,including proteoglycans, such as small leucine rich proteoglycans(SLRPs). In some embodiments, the SLRP is decorin, biglycan and/orosteoadherin. As described herein, the complexes also differ in contentwith respect to the presence of particular sulfated glycosaminoglycans,such keratan sulfate. In addition, as described herein, complexesisolated from AM or UC using different extraction methods (e.g., PBSversus GnHCl extraction) resulted in complexes with differentbiochemical compositions and biological properties. In certaininstances, it is found that complexes isolated from an insolublefraction by GnHCl extraction from umbilical cord tissue exhibit improvedproperties.

PTX3 is a multimeric protein that has been shown to interact directlywith TSG-6 and IαI HCs. PTX3 is upregulated in response to inflammatoryregulators and has been shown to play an important role in theorganization of HA in the extracellular matrix of the cumulous oophorousduring oocyte maturation. As demonstrated herein, PTX3 also is foundwithin nHC-HA complexes (i.e. nHC-HA/PTX3) of the amniotic membrane andumbilical cord and plays a critical role in M2 macrophage polarization.

M1 macrophages, or classically activated proinflammatory macrophages areinduced by interferon (IFN) alone or in combination withlipopolysaccharide (LPS) or tumor necrosis factor (TNF) α. M1macrophages are typically characterized by high expression ofinterleukin-12 (IL-12) and IL-23 and low levels of IL-10. In contrast,M2 macrophages or “alternatively activated” macrophages display woundhealing and tissue regenerative properties and are characterized by lowIL-12/IL-23 and high IL-10 or about the same ratio of IL-12 to IL-10. Incertain instances, M2 macrophages also have a high expression of TGF-β.

The examples provided herein demonstrate that PTX3 binds directly toimmobilized HA as evidenced by resistance to dissociating agents. It isdemonstrated herein that in vitro reconstituted complexes of HA bound toPTX3 exhibit different properties compared to in vitro reconstitutedcomplexes of HA bound to TSG-6. For example, in some embodiments, aPTX3/HA complex promotes attachment of LPS-stimulated macrophageswithout aggregation and induces expression of IL-10 in an LPS-stimulatedmacrophage. In some embodiments, a PTX3/HA complex disclosed hereinincreases the expression of IL-10 by about 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%in an LPS-stimulated macrophage compared to IL-10 expression the absenceof a PTX3/HA complex. In contrast, in some embodiments, the TSG-6/HAcomplex reduces cell attachment and promotes aggregation ofLPS-stimulated macrophages and does not induce the expression of IL-10in an LPS-stimulated macrophage. In addition, in some embodiments, TSG-6pre-bound to HA inhibits subsequent binding of PTX3 to the complex. Insome embodiments, both TSG-6/HA complex and PTX3/HA complex decreasedexpression of IL-12 in an LPS-stimulated macrophage. In someembodiments, a PTX3/HA complex or TSG-6/HA complex disclosed hereinreduces or inhibits the expression of IL-12 by about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% in an LPS-stimulated macrophage compared to IL-12expression the absence of a PTX3/HA complex or TSG-6/HA complex. In someembodiments, both TSG-6/HA complex and PTX3/HA complex increasedexpression of IL-23 in an LPS/IFNγ-stimulated macrophage. In someembodiments, a PTX3/HA complex or TSG-6/HA complex disclosed hereinreduces or inhibits the expression of IL-23 by about 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% in an LPS/IFNγ-stimulated macrophage compared to IL-23expression the absence of a PTX3/HA complex or TSG-6/HA complex.

In addition, it is demonstrated herein that in vitro reconstitutedrcHC-HA/PTX3 complexes possess different biological activities dependenton whether the rcHC-HA/PTX3 complex is formed with HA pre-bound to TSG-6in the presence of IαI followed by addition of PTX3 or HA pre-bound toPTX3 followed by addition of TSG-6 with IαI. Exemplary methods forreconstitution of rcHC-HA/PTX3 complexed formed with HA pre-bound toTSG-6 or HA pre-bound to PTX3 are provided herein. In some embodiments,rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to TSG-6result in aggregation of LPS-stimulated macrophages. In someembodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-boundto PTX3 promote attachment of LPS-stimulated macrophages withoutaggregation.

In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HApre-bound to PTX3 decrease or inhibit expression of M1 macrophagemarkers such as IL-12 and IL-23. In some embodiments, rcHC-HA/PTX3complexes formed with immobilized HA pre-bound to PTX3 decreaseexpression of IL-12 in an LPS-stimulated macrophage compared to IL-12expression the absence of rcHC-HA/PTX3 complex. In some embodiments, anrcHC-HA/PTX3 complex disclosed herein reduces or inhibits the expressionof IL-12 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in an LPS-stimulatedmacrophage compared to IL-12 expression the absence of rcHC-HA/PTX3complex. In some embodiments, rcHC-HA/PTX3 complexes formed withimmobilized HA pre-bound to PTX3 decrease or inhibit expression of IL-23in an LPS/IFNγ-stimulated macrophage compared to IL-12 expression theabsence of rcHC-HA/PTX3 complex. In some embodiments, rcHC-HA/PTX3complexes formed with immobilized HA pre-bound to PTX3 decrease orinhibit expression of IL-23 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% inan LPS/IFNγ-stimulated macrophage compared to IL-23 expression theabsence of rcHC-HA/PTX3 complex. In some embodiments, rcHC-HA/PTX3complexes formed with immobilized HA pre-bound to PTX3 replicate theactivity of nHC-HA/PTX3 complexes isolated from amniotic membrane.

In some embodiments, rcHC-HA/PTX3 complexes formed with immobilized HApre-bound to TSG-6 decrease or inhibit expression of M1 macrophagemarkers such as IL-12, but increase expression of IL-23. In someembodiments, rcHC-HA/PTX3 complexes formed with immobilized HA pre-boundto TSG-6 decrease or inhibit expression of IL-12. In some embodiments,rcHC-HA/PTX3 complexes formed with immobilized HA pre-bound to TSG-6increase expression of IL-23.

Provided herein are methods of producing reconstituted HC-HA/PTX3complexes using immobilized HA pre-bound to PTX3 and uses thereof. Alsoprovided herein are complexes of immobilized HA pre-bound to PTX3 anduses thereof. Also provided herein are methods of producingreconstituted HC-HA/PTX3 complexes using immobilized HA pre-bound toTSG-6 and uses thereof. In some embodiments, the reconstitutedHC-HA/PTX3 complexes provided herein are administered to treat a widevariety of diseases or conditions, including, but not limited to, thetreatment, such as the inhibition, reduction, prevention or lowering therisk, of inflammation, immune reaction leading to autoimmune or immunerejection, adhesion, scarring, angiogenesis, conditions requiring cellor tissue regeneration, tissue reperfusion injury due to ischemia,including myocardial infarction and stroke, and the symptoms causedthereby. In some embodiments, the reconstituted HC-HA/PTX3 complexesprovided herein are administered to treat inflammation. In someembodiments, the reconstituted HC-HA/PTX3 complexes provided herein areadministered to treat scarring. In some embodiments, the reconstitutedHC-HA/PTX3 complexes provided herein are administered to treatangiogenesis. In some embodiments, the reconstituted HC-HA/PTX3complexes provided herein are administered to treat immune reactionleading to autoimmune or immune rejection. In some embodiments, thereconstituted HC-HA/PTX3 complexes provided herein are administered totreat conditions requiring inhibition of cell adhesion. In someembodiments, the reconstituted HC-HA/PTX3 complexes provided herein areadministered to treat conditions requiring cell or tissue regeneration.

In addition, the examples provided herein demonstrate the ability ofHC-HA/PTX3 complexes to maintain stem cells in an undifferentiated stateas well as induce adult differentiated fibroblasts to youngerprogenitors in a human corneal fibroblasts model. Human cornealfibroblasts are differentiated from keratocytes and upon addition ofexogenous TGF-β1, they further differentiate into scar-formingmyofibroblasts. The data provided herein demonstrate that culturing thecells in the presence of HC-HA prevented cells from differentiating intomyofibroblasts under TGF-β1 stimulation. In the absence of TGF-β1,HC-HA/PTX3 complexes revert human corneal fibroblasts into keratocytesexpressing keratocan and CD34. In the presence of TGF-β1 human cornealfibroblasts are further reprogrammed into younger progenitors that lackof keratocan expression and but express a number of neural crest cellmarkers such as Osr2, FGF10, and Sox9 and embryonic stem cell markers,such as c-myc, KLF4, Nanog, nestin, Oct 4, Rex-1, Sox-2, and SSEA-4.

The transcription factors Sox2, Oct4, c-Myc, and KLF4 are known play animportant role in the induction of progenitor stem cells (iPSCs) fromadult differentiated cells. Accordingly, in some embodiments, HC-HA/PTX3complexes provided herein are employed to reprogram adult differentiatedcells into iPSCs. In some embodiments, induction of iPSCs using anHC-HA/PTX3 complex in combination with one or more of Sox2, Oct4, c-Myc,and KLF4 is performed with a much higher efficiency than theconventional methods that use these four transcription factors withoutHC-HA/PTX3. In some embodiments, addition of HC-HA/PTX3 complexfacilitates stem cell induction by turning off TGF-β signaling toprevent differentiation and by turning on BMP signaling to facilitatereprogramming into young progenitor cells such as iPSCs. In someembodiments, addition of HC-HA/PTX3 complex facilitates stem cellinduction by reprogramming cells into younger progenitors and inductionof stem cell markers. In some embodiments, addition of HC-HA/PTX3complex helps maintain stem cell characteristics during ex vivoexpansion, thus eliminating the need of using feeder layers made ofmurine embryonic fibroblasts. Hence, in some embodiments, HC-HA/PTX3complex is used as a carrier or scaffold to help deliver stem cells thathave been ex vivo expanded into the human patients to promote theefficacy of said stem cell therapies.

Methods of Production of Isolated nHC-HA/PTX3 Complexes

Disclosed herein are methods of generating isolated native HC-HA/PTX3complexes (nHC-HA/PTX3).

In some embodiments, the isolated nHC-HA/PTX3 complex is isolated froman amniotic tissue. In some embodiments, the isolated nHC-HA/PTX3complex is isolated from an amniotic membrane or an umbilical cord. Insome embodiments, the isolated nHC-HA/PTX3 complex is isolated fromfresh, frozen or previously frozen placental amniotic membrane (PAM),fresh, frozen or previously frozen umbilical cord amniotic membrane(UCAM), fresh, frozen or previously frozen placenta, fresh, frozen orpreviously frozen umbilical cord, fresh, frozen or previously frozenchorion, fresh, frozen or previously frozen amnion-chorion, or anycombinations thereof. Such tissues can be obtained from any mammal, suchas, for example, but not limited to a human, non-human primate, cow orpig.

In some embodiments, the nHC-HA/PTX3 is purified by any suitable method.In some embodiments, the nHC-HA/PTX3 complex is purified bycentrifugation (e.g., ultracentrifugation, gradient centrifugation),chromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), gel filtration,, or differentialsolubility, ethanol precipitation or by any other available techniquefor the purification of proteins (See, e.g., Scopes, ProteinPurification Principles and Practice 2nd Edition, Springer-Verlag, NewYork, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression:A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P.,Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification:Methods in Enzymology (Methods in Enzymology Series, Vol 182), AcademicPress, 1997, all incorporated herein by reference).

In some embodiments, the nHC-HA/PTX3 is isolated from an extract. Insome embodiments, the extract is prepared from an amniotic membraneextract. In some embodiments, the extract is prepared from an umbilicalcord extract. In some embodiments, the umbilical cord extract comprisesumbilical cord stroma and/or Wharton’s jelly. In some embodiments, thenHC-HA/PTX3 complex is contained in an extract that is prepared byultracentrifugation. In some embodiments, the nHC-HA/PTX3 complex iscontained in an extract that is prepared by ultracentrifugation using aCsCl/4-6M guanidine HCl gradient. In some embodiments, the extract isprepared by at least 2 rounds of ultracentrifugation. In someembodiments, the extract is prepared by more than 2 rounds ofultracentrifugation (i.e. nHC-HA/PTX3 2^(nd)). In some embodiments, theextract is prepared by at least 4 rounds of ultracentrifugation (i.e.nHC-HA/PTX3 4^(th)). In some embodiments, the nHC-HA/PTX3 complexcomprises a small leucine-rich proteoglycan. In some embodiments, thenHC-HA/PTX3 complex comprises HC1, HA, PTX3 and/or a small leucine-richproteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by extraction in an isotonic solution. In some embodiments, theisotonic solution is PBS. For example, in some embodiments the tissue ishomogenized in PBS to produce a homogenized sample. The homogenizedsample is then separated into a soluble portion and insoluble portion bycentrifugation. In some embodiments, ultracentrifugation is performed onthe soluble portion of the PBS-extracted tissue. In such embodiments,the nHC-HA/PTX3 purified by ultracentrifugation of the PBS-extractedtissue called an nHC-HA/PTX3 soluble complex. In some embodiments, thenHC-HA soluble complex comprises a small leucine-rich proteoglycan. Insome embodiments, the nHC-HA/PTX3 soluble complex comprises HC1, HA,PTX3 and/or a small leucine-rich proteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by direct guanidine HCl extraction (e.g. 4-6 M GnHCl) of theamniotic membrane and/or umbilical cord tissue. In some embodiments, theGnHCl extract tissues is then centrifuged to produce GnHCl soluble andGnHCl insoluble portions. In some embodiments, ultracentrifugation isperformed on the GnHCl soluble portion. In such embodiments, thenHC-HA/PTX3 purified by ultracentrifugation of the guanidineHCl-extracted tissue is called an nHC-HA/PTX3 insoluble complex. In someembodiments, the nHC-HA insoluble complex comprises a small leucine-richproteoglycan. In some embodiments, the nHC-HA/PTX3 insoluble complexcomprises HC1, HA, PTX3 and/or a small leucine-rich proteoglycan.

In some embodiments, ultracentrifugation is performed on an extractprepared by further guanidine HCl extraction of the insoluble portion ofthe PBS-extracted tissue. For example, in some embodiments the tissue ishomogenized in PBS to produce a homogenized sample. The homogenizedsample is then separated into a soluble portion and insoluble portion bycentrifugation. The insoluble portion is then further extracted inguanidine HCl (e.g. 4-6 M GnHCl) and centrifuged to produce a guanidineHCl soluble and insoluble portions. In some embodiments,ultracentrifugation is performed on the guanidine HCl soluble portion.In such embodiments, the nHC-HA/PTX3 purified by ultracentrifugation ofthe guanidine HCl-extracted tissue is called an nHC-HA/PTX3 insolublecomplex. In some embodiments, the nHC-HA insoluble complex comprises asmall leucine-rich proteoglycan. In some embodiments, the nHC-HA/PTX3insoluble complex comprises HC1, HA, PTX3 and/or a small leucine-richproteoglycan.

In some embodiments, the method of purifying the isolated nHC-HA/PTX3extract comprises: (a) dissolving the isolated extract (e.g. prepared bythe soluble or insoluble method described herein) in CsCl/4-6M guanidineHCl at the initial density of 1.35 g/ml, to generate a CsCl mixture, (b)centrifuging the CsCl mixture at 125,000 x g for 48 h at 15° C., togenerate a first purified extract, (c) extracting the first purifiedextract and dialyzing it against distilled water to remove CsCl andguanidine HCl, to generate a dialysate. In some embodiments, the methodof purifying the isolated extract further comprises (d) mixing thedialysate with 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v)potassium acetate at 0° C. for 1 h, to generate a firstdialysate/ethanol mixture, (e) centrifuging the first dialysate/ethanolmixture at 15,000 x g, to generate a second purified extract, and (f)extracting the second purified extract. In some embodiments, the methodof purifying the isolated extract further comprises: (g) washing thesecond purified extract with ethanol (e.g., 70% ethanol), to generate asecond purified extract/ethanol mixture; (h) centrifuging the secondpurified extract/ethanol mixture, to generate a third purified extract;and (i) extracting the third purified extract. In some embodiments, themethod of purifying the isolated extract further comprises: (j) washingthe third purified extract with ethanol (e.g., 70% ethanol), to generatea third purified extract/ethanol mixture; (k) centrifuging the thirdpurified extract/ethanol mixture, to generate a forth purified extract;and (l) extracting the forth purified extract. In some embodiments, thepurified extract comprises an nHC-HA/PTX3 complex.

In some embodiments, the nHC-HA/PTX3 complex is purified byimmunoaffinity chromatography. In some embodiments, anti HC1 antibodies,anti-HC2 antibodies, or both are generated and affixed to a stationarysupport. In some embodiments, the unpurified HC-HA complex (i.e., themobile phase) is passed over the support. In certain instances, theHC-HA complex binds to the antibodies (e.g., via interaction of (a) ananti-HC1 antibody and HC1, (b) an anti-HC2 antibody and HC2, (c) ananti-PTX antibody and PTX3, (d) an anti-SLRP antibody and the SLRP, or(e) any combination thereof). In some embodiments the support is washed(e.g., with PBS) to remove any unbound or loosely bound molecules. Insome embodiments, the support is then washed with a solution thatenables elution of the nHC-HA/PTX3 complex from the support (e.g., 1%SDS, 6M guanidine-HCl, or 8M urea).

In some embodiments, the nHC-HA/PTX3 complex is purified by affinitychromatography. In some embodiments, HABP is generated and affixed to astationary support. In some embodiments, the unpurified nHC-HA/PTX3complex (i.e., the mobile phase) is passed over the support. In certaininstances, the nHC-HA/PTX3 complex binds to the HABP. In someembodiments the support is washed (e.g., with PBS) to remove any unboundor loosely bound molecules. In some embodiments, the support is thenwashed with a solution that enables elution of the HC-HA complex fromthe support.

In some embodiments, the nHC-HA/PTX3 complex is purified by acombination of HABP affinity chromatography, and immunoaffinitychromatography using anti HC1 antibodies, anti-HC2 antibodies, anti-PTX3antibodies, antibodies against a SLRP or a combination of SLRPs, or anycombination of antibodies thereof.

In some embodiments, the nHC-HA/PTX3 complex is purified from theinsoluble fraction as described herein using one or more antibodies. Insome embodiments, the nHC-HA/PTX3 complex is purified from the insolublefraction as described herein using anti-SLRP antibodies.

In some embodiments, the nHC-HA/PTX3 complex is purified from thesoluble fraction as described herein. In some embodiments, thenHC-HA/PTX3 complex is purified from the soluble fraction as describedherein using anti-PTX3 antibodies.

In some embodiments, the nHC-HA/PTX3 complex comprises a small leucinerich proteoglycan (SLRP). In some embodiments, the nHC-HA/PTX3 complexcomprises a class I, class II or class II SLRP. In some embodiments, thesmall leucine-rich proteoglycan is selected from among class I SLRPs,such as decorin and biglycan. In some embodiments, the smallleucine-rich proteoglycan is selected from among class II SLRPs, such asfibromodulin, lumican, PRELP (proline arginine rich end leucine-richprotein), keratocan, and osteoadherin. In some embodiments, the smallleucine-rich proteoglycan is selected from among class III SLRPs, suchas epipycan and osteoglycin. In some embodiments, the small leucine-richproteoglycan is selected from among bikunin, decorin, biglycan, andosteoadherin. In some embodiments, the small leucine-rich proteincomprises a glycosaminoglycan. In some embodiments, the smallleucine-rich proteoglycan comprises keratan sulfate.

Methods of Production of rcHC-HA/PTX3 Complexes

Disclosed herein are methods of generating reconstituted HC-HA/PTX3complexes (rcHC-HA/PTX3) with or without SLRPs. Also disclosed hereinare rcHC-HA/PTX3 complexes and intermediate combinations of componentsgenerated by such methods.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with pentraxin 3 (PTX3) under suitable conditions toform a PTX3/HA complex, and (b) contacting the PTX3/HA complex with IαIand Tumor necrosis factor-Stimulated Gene-6 (TSG-6). Provided herein arercHC-HA/PTX3 complexes produced by such method. In some embodiments,TSG-6 catalyzes the transfer of heavy chain 1 (HC1) of inter-α-inhibitor(IαI) to HA. In some embodiments, HC1 of IαI forms a covalent linkagewith HA. In some embodiments, the steps (a) and (b) of the method areperformed sequentially in order.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises contacting a PTX3/HA complex with IαI and TSG-6. Insome embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HC1) ofinter-α-inhibitor (IαI) to HA. Provided herein are rcHC-HA/PTX3complexes produced by such method. In some embodiments, HC1 of IαI formsa covalent linkage with HA.

In some embodiments, a method for generating a complex of HA bound toPTX3 comprises contacting immobilized high molecular weight hyaluronan(HMW HA) with pentraxin 3 (PTX3) under suitable conditions to form aPTX3/HA complex. Provided herein are PTX3/HA complexes produced by suchmethod.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with IαI and TSG-6 to HA to form an HC-HA complexpre-bound to TSG-6 and (b) contacting the HC-HA complex with pentraxin 3(PTX3) under suitable conditions to form an rcHC-HA/PTX3 complex.Provided herein are rcHC-HA/PTX3 complexes produced by such method. Insome embodiments, HC1of IαI forms a covalent linkage with HA. In someembodiments, the steps (a) and (b) of the method are performedsequentially in order. In some embodiments, the method comprisescontacting an HC-HA complex pre-bound to TSG-6 with PTX3.

In some embodiments, the method comprises first contacting highmolecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, then contacting thePTX3/HA complex with IαI and TSG-6.

In some embodiments, the IαI protein and TSG-6 protein are contacted tothe complex at a molar ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1,8:1, 9:1, 10:1, 15: 1, or 20:1 (IαI:TSG-6). In some embodiments theratio of IαI: TSG-6 ranges from about 1:1 to about 20:1, such as about1:1 to about 10:1, such as about 1: 1 to 5 about:1, such as about 1:1 toabout 3: 1. In some embodiments, the ratio of IαI:TSG-6 is 3:1 orhigher. In some embodiments, the ratio of IαI:TSG-6 is 3: 1.

In some embodiments, the steps (a) and (b) of the method are performedsequentially in order. In some embodiments, the method comprisescontacting a PTX3/HA complex with IαI and TSG-6.

In certain instances, TSG-6 interacts with IαI and forms covalentcomplexes with HC1 and HC2 of IαI (i.e. HC1•TSG-6 and HC2•TSG-6). Incertain instances, in the presence of HA, the HCs are transferred to HAto form rcHC-HA. In some embodiments, a TSG-6•HC1 complex is added topre-bound PTX3/HA complex to catalyze the transfer of HC1 to HA. In someembodiments, the method comprises first contacting immobilized highmolecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, then contacting thePTX3/HA complex with a HC1•TSG-6 complex. In some embodiments, acombination of HC1•TSG-6 complex and HC2•TSG-6 complex is added to aPTX3/HA complex.

In some embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs for at least 10 minutes, at least 30 minutes, at least 1 hour, atleast 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, atleast 6 hours, at least 12 hours, or at least 24 hours or longer. Insome embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs for at least 2 hours or longer. In some embodiments, the step ofcontacting PTX3 to immobilized HMW HA occurs for at least 2 hours. Insome embodiments, the step of contacting PTX3 to immobilized HMW HAoccurs at 37° C. In some embodiments, the step of contacting PTX3 toimmobilized HMW HA occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the step of contacting the PTX3/HA complex with IαIand TSG-6 to HA occurs for at least 10 minutes, at least 30 minutes, atleast 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, atleast 5 hours, at least 6 hours, at least 12 hours, or at least 24 hoursor longer. In some embodiments the step of contacting the PTX3/HAcomplex with a HC1•TSG-6 complex and/or a HC2•TSG-6 complex occurs forat least 10 minutes, at least 30 minutes, at least 1 hour, at least 2hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6hours, at least 12 hours, or at least 24 hours or longer. In someembodiments the step of contacting the PTX3/HA complex with a HC1•TSG-6complex and/or a HC2•TSG-6 complex occurs for at least 2 hours orlonger. In some embodiments the step of contacting the PTX3/HA complexwith a HC1•TSG-6 complex and/or a HC2•TSG-6 complex occurs for at least2 hours. In some embodiments the step of contacting the PTX3/HA complexwith a HC1•TSG-6 complex and/or a HC1•TSG-6 complex occurs at 37° C. Insome embodiments the step of contacting the PTX3/HA complex with aHC1•TSG-6 complex and/or a HC1•TSG-6 complex occurs in 5 mM MgCl₂ inPBS.

In some embodiments, the method comprises contacting high molecularweight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein,inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and Tumornecrosis factor α-stimulated gene 6 (TSG-6) simultaneously undersuitable conditions to form a HC-HA/PTX3 complex. In some embodiments,the contacting the HMW HA with PTX3, IαI and TSG-6 occurs for at least10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, atleast 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, atleast 12 hours, or at least 24 hours or longer. In some embodiments thestep of contacting the HMW HA, PTX3, IαI, and TSG-6 occurs at 37° C. Insome embodiments the step of contacting the HMW HA, PTX3, IαI, and TSG-6occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the method comprises contacting high molecularweight hyaluronan (HMW HA) with a pentraxin 3 (PTX3) protein,inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and Tumornecrosis factor α-stimulated gene 6 (TSG-6) sequentially, in any order,under suitable conditions to form a HC-HA/PTX3 complex. In someembodiments, the contacting the HMW HA with PTX3, IαI and TSG-6 occursfor at least 10 minutes, at least 30 minutes, at least 1 hour, at least2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least6 hours, at least 12 hours, or at least 24 hours or longer. In someembodiments the step of contacting the HMW HA, PTX3, IαI, and TSG-6occurs at 37° C. In some embodiments the step of contacting the HMW HA,PTX3, IαI, and TSG-6 occurs in 5 mM MgCl₂ in PBS.

In some embodiments, the methods for production of an rcHC-HA/PTX3complex further comprises addition of one or more small leucine richproteoglycans (SLRPs). In some embodiments, a method for generatingreconstituted HC-HA/PTX3 complexes comprises (a) contacting immobilizedhigh molecular weight hyaluronan (HMW HA) with pentraxin 3 (PTX3) undersuitable conditions to form a PTX3/HA complex, (b) contacting thePTX3/HA complex with IαI and Tumor necrosis factor-Stimulated Gene-6(TSG-6) and (c) contacting the PTX3/HA complex with one or more SLRPS.Provided herein are rcHC-HA/PTX3 complexes produced by such method. Insome embodiments, TSG-6 catalyzes the transfer of heavy chain 1 (HC1) ofinter-α-inhibitor (IαI) to HA. In some embodiments, HC1 of IαI forms acovalent linkage with HA. In some embodiments, the steps (a), (b), and(c) of the method are performed sequentially in order. In someembodiments, the steps (a), (b), and (c) of the method are performedsimultaneously. In some embodiments, the step (a) of the method isperformed and then steps (b) and (c) of the method are performedsequentially in order. In some embodiments, the step (a) of the methodis performed and then steps (b) and (c) of the method are performedsimultaneously.

In some embodiments, a method for generating reconstituted HC-HA/PTX3complexes comprises (a) contacting immobilized high molecular weighthyaluronan (HMW HA) with IαI and TSG-6 to HA to form an HC-HA complexpre-bound to TSG-6, (b) contacting the HC-HA complex with pentraxin 3(PTX3) and (c) contacting the HC-HA complex with one or more SLRPS undersuitable conditions to form an rcHC-HA/PTX3 complex. Provided herein arercHC-HA/PTX3 complexes produced by such method. In some embodiments,HC1of IαI forms a covalent linkage with HA. In some embodiments, themethod comprises contacting an HC-HA complex pre-bound to TSG-6 withPTX3. In some embodiments, the steps (a), (b), and (c) of the method areperformed sequentially in order. In some embodiments, the steps (a),(b), and (c) of the method are performed simultaneously. In someembodiments, the step (a) of the method is performed and then steps (b)and (c) of the method are performed sequentially in order. In someembodiments, the step (a) of the method is performed and then steps (b)and (c) of the method are performed simultaneously.

In some embodiments, the SLRP is selected from among a class I, class IIor class II SLRP. In some embodiments, the SLRP is selected from amongclass I SLRPs, such as decorin and biglycan. In some embodiments, thesmall leucine-rich proteoglycan is selected from among class II SLRPs,such as fibromodulin, lumican, PRELP (proline arginine rich endleucine-rich protein), keratocan, and osteoadherin. In some embodiments,the small leucine-rich proteoglycan is selected from among class IIISLRPs, such as epipycan and osteoglycin. In some embodiments, the smallleucine-rich proteoglycan is selected from among bikunin, decorin,biglycan, and osteoadherin. In some embodiments, the small leucine-richprotein comprises a glycosaminoglycan. In some embodiments, the smallleucine-rich proteoglycan comprises keratan sulfate.

PTX3

In some embodiments, PTX3 for use in the methods is isolated from a cellor a plurality of cells (e.g., a tissue extract). Exemplary cellssuitable for the expression of PTX3 include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, PTX3 foruse in the methods is isolated from a human cell. In some embodiments,PTX3 for use in the methods is isolated from a cell that is stimulatedwith one or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic membrane cell. In some embodiments, PTX3 for use in the methodsis isolated from an amniotic membrane cell from an umbilical cord. Insome embodiments, the amniotic membrane cell is stimulated with or moreproinflammatory cytokines to upregulate PTX3 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anumbilical cord cell. In some embodiments, the umbilical cord cell isstimulated with or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic epithelial cell. In some embodiments, PTX3 for use in themethods is isolated from an umbilical cord epithelial cell. In someembodiments, the amniotic epithelial cell or umbilical cord epithelialcell is stimulated with or more proinflammatory cytokines to upregulatePTX3 expression. In some embodiments, the proinflammatory cytokine isIL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is isolated from anamniotic stromal cell. In some embodiments, PTX3 for use in the methodsis isolated from an umbilical cord stromal cell. In some embodiments,the amniotic stromal cell or umbilical cord stromal cell is stimulatedwith or more proinflammatory cytokines to upregulate PTX3 expression. Insome embodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 for use in the methods is a native PTX3protein isolated from a cell. In some embodiments, the cell isstimulated with or more proinflammatory cytokines to upregulate PTX3expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, PTX3 is prepared by recombinant technology. In someembodiments, PTX3 is expressed from a recombinant expression vector. Insome embodiments, nucleic acid encoding PTX3 is operably linked to aconstitutive promoter. In some embodiments, nucleic acid encoding PTX3is operably linked to an inducible promoter. In some embodiments, PTX3is expressed in a transgenic animal. In some embodiments, PTX3 is arecombinant protein. In some embodiments, PTX3 is a recombinant proteinisolated from a cell. In some embodiments, PTX3 is a recombinant proteinproduced in a cell-free extract.

In some embodiments, PTX3 is purified from amniotic membrane, umbilicalcord, umbilical cord amniotic membrane, chorionic membrane, amnioticfluid, or a combination thereof. In some embodiments, PTX3 is purifiedfrom amniotic membrane cells. In some embodiments, the amniotic membranecell is an amniotic epithelial cell. In some embodiments, the amnioticmembrane cell is an umbilical cord epithelial cell. In some embodiments,the amniotic membrane cell is an amniotic stromal cell. In someembodiments, the amniotic membrane cell is an umbilical cord stromalcell. In some embodiments, the amniotic membrane cell is stimulated withor more proinflammatory cytokines to upregulate PTX3 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, PTX3 is not isolated from a cell or a plurality ofcells (e.g., a tissue extract).

In some embodiments, PTX3 comprises a polypeptide having the sequenceset forth in SEQ ID NO: 33 or a variant thereof having at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:33. Exemplary variants include, for example, species variants, allelicvariants and variants that contain conservative and non-conservativeamino acid mutations. In some embodiments, PTX3 comprises a fragment ofPTX3 sufficient to bind to HA and facilitate the formation ofrcHC-HA/PTX3 complex. In some embodiments, PTX3 comprises Glu18 toSer277 of human PTX3. Variants of PTX3for use in the provided methodsinclude variants with an amino acid modification that is an amino acidreplacement (substitution), deletion or insertion. In some embodiments,such modification improves one or more properties of the PTX3polypeptides such as improving the one or more therapeutic properties ofthe rcHC-HA/PTX3 complex (e.g., antiinflammatory, anti-immune,anti-angiogenic, anti-scarring, anti-adhesion, regeneration or othertherapeutic activities as described herein).

In some embodiments PTX3 protein is obtained from a commercial source.An exemplary commercial source for PTX3 is, but is not limited to, PTX3(Catalog No. 1826-TS; R&D Systems, Minneapolis, MN).

In some embodiments, the PTX3 protein used in the methods is amultimeric protein. In some embodiments, the PTX3 protein used in themethods is a homomultimer. In some embodiments, the homomultimer is adimer, trimer, tetramer, hexamer, pentamer, or octamer. In someembodiments, the PTX3 homomultimer is a trimer, tetramer, or octamer. Inparticular embodiments, the PTX3 homomultimer is an octamer. In someembodiments, the multimerization domain is modified to improvemultimerization of the PTX3 protein. In some embodiments, themultimerization domain is replaced with a heterogeneous multimerizationdomain (e.g., an Fc multimerization domain or leucine zipper) that whenfused to PTX3 improves the multimerization of PTX3.

TSG-6

In some embodiments, TSG-6 for use in the methods is isolated from acell or a plurality of cells (e.g., a tissue extract). Exemplary cellssuitable for the expression of TSG-6 include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, TSG-6for use in the methods is isolated from a human cell. In someembodiments, TSG-6 for use in the methods is isolated from a cell thatis stimulated with one or more proinflammatory cytokines to upregulateTSG-6 expression. In some embodiments, the proinflammatory cytokine isIL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic membrane cell. In some embodiments, TSG-6 for use in themethods is isolated from an amniotic membrane cell from an umbilicalcord. In some embodiments, TSG-6 for use in the methods is isolated froman amniotic membrane cell that is stimulated with one or moreproinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anumbilical cord cell. In some embodiments, TSG-6 for use in the methodsis isolated from an umbilical cord cell that is stimulated with one ormore proinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic epithelial cell. In some embodiments, TSG-6 for use in themethods is isolated from an umbilical cord epithelial cell. In someembodiments, TSG-6 for use in the methods is isolated from an amnioticepithelial cell or an umbilical cord epithelial cell that is stimulatedwith one or more proinflammatory cytokines to upregulate TSG-6expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, TSG-6 for use in the methods is isolated from anamniotic stromal cell. In some embodiments TSG-6 for use in the methodsis isolated from an umbilical cord stromal cell. In some embodiments,TSG-6 for use in the methods is isolated from an amniotic stromal cellor an umbilical cord stromal cell that is stimulated with one or moreproinflammatory cytokines to upregulate TSG-6 expression. In someembodiments, the proinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 for use in the methods is a native TSG-6protein isolated from a cell. In some embodiments, the cell isstimulated with or more proinflammatory cytokines to upregulate TSG-6expression. In some embodiments, the proinflammatory cytokine is IL-1 orTNF-α.

In some embodiments, TSG-6 is prepared by recombinant technology. Insome embodiments, TSG-6 is expressed from a recombinant expressionvector. In some embodiments, nucleic acid encoding TSG-6 is operablylinked to a constitutive promoter. In some embodiments, nucleic acidencoding TSG-6 is operably linked to an inducible promoter. In someembodiments, TSG-6 is expressed in a transgenic animal. In someembodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6is a recombinant protein isolated from a cell. In some embodiments,TSG-6 is a recombinant protein produced in a cell-free extract.

In some embodiments, TSG-6 is purified from amniotic membrane, amnioticmembrane, chorionic membrane, amniotic fluid, or a combination thereof.In some embodiments, PTX3 is purified from amniotic membrane cells. Insome embodiments, the amniotic membrane cell is an amniotic epithelialcell. In some embodiments, the amniotic epithelial cell is an umbilicalcord epithelial cell. In some embodiments, the amniotic membrane cell isan amniotic stromal cell. In some embodiments, the amniotic membranecell is an umbilical cord stromal cell. In some embodiments, theamniotic membrane cell is stimulated with or more proinflammatorycytokines to upregulate TSG-6 expression. In some embodiments, theproinflammatory cytokine is IL-1 or TNF-α.

In some embodiments, TSG-6 is not isolated from a cell or a plurality ofcells (e.g., a tissue extract).

In some embodiments, TSG-6 comprises a fragment of TSG-6 that issufficient to facilitate or catalyze the transfer HC1 of IαI to HA. Insome embodiments, TSG-6 comprises the link module of TSG-6. In someembodiments, TSG-6 comprises amino acids Trp18 through Leu277 of TSG-6.In some embodiments, TSG-6 comprises a polypeptide having the sequenceset forth in SEQ ID NO: 2 or a variant thereof having at least 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in SEQ ID NO:2. Exemplary variants include, for example, species variants, allelicvariants and variants that contain conservative and non-conservativeamino acid mutations. Natural allelic variants of human TSG-6 include,for example, TSG-6 containing the amino acid replacement Q144R. Variantsof TSG-6 or HA binding fragments thereof for use in the provided methodsinclude variants with an amino acid modification that is an amino acidreplacement (substitution), deletion or insertion. In some embodiments,such modification improve one or more properties of the TSG-6polypeptides such as improved transfer of HC1 of IαI to HA or improvedrelease of the TSG-6 polypeptide from the rcHC-HA/PTX3 complex followingtransfer of HC1 of IαI to HA.

In some embodiments, TSG-6 comprises an affinity tag. Exemplary affinitytags include but are not limited to a hemagglutinin tag, apoly-histidine tag, a myc tag, a FLAG tag, a glutathione-S-transferase(GST) tag. Such affinity tags are well known in the art for use inpurification. In some embodiments, such an affinity tag incorporatedinto the TSG-6 polypeptide as a fusion protein or via a chemical linker.In some embodiments, TSG-6 comprises an affinity tag and the unboundTSG-6 is removed from the rcHC-HA/PTX3 complex by affinity purification.

In some embodiments TSG-6 protein is obtained from a commercial source.An exemplary commercial source for TSG-6 is, but is not limited to,TSG-6 (Catalog No. 2104-TS R&D Systems, Minneapolis, MN).

IαI

In some embodiments, the IαI comprises an HC1 chain. In someembodiments, the IαI comprises an HC1 and an HC2 chain. In someembodiments, the IαI comprises an HC1 and bikunin. In some embodiments,the IαI comprises an HC1, and HC2 chain and bikunin. In someembodiments, the IαI comprises an HC1, and HC2 chain and bikunin linkedby a chondroitin sulfate chain.

In some embodiments, IαI is isolated from a biological sample. In someembodiments the biological sample is a biological sample from a mammal.In some embodiments, the mammal is a human. In some embodiments, thebiological sample is a blood, serum, plasma, liver, amniotic membrane,chorionic membrane or amniotic fluid sample. In some embodiments, thebiological sample is a blood, serum, or plasma sample. In someembodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the IαI is purified from human blood, plasma or serum. Insome embodiments, IαI is isolated from human serum. In some embodiments,IαI is not isolated from serum. In some embodiments, IαI for use in themethods is produced in an amniotic membrane cell. In some embodiments,IαI for use in the methods is produced in an umbilical cord cell. Insome embodiments, IαI for use in the methods is produced in an amnioticmembrane cell from an umbilical cord. In some embodiments, IαI for usein the methods is produced in an amniotic epithelial cell. In someembodiments, IαI for use in the methods is produced in an umbilical cordepithelial cell. In some embodiments, IαI for use in the methods isproduced in an amniotic stromal cell. In some embodiments, IαI for usein the methods is produced in an umbilical cord stromal cell. In someembodiments, IαI for use in the methods is produced in a hepatic cell.In some embodiments, IαI is prepared by recombinant technology.

In some embodiments, HC1 of IαI is isolated from a biological sample. Insome embodiments the biological sample is a biological sample from amammal. In some embodiments, the mammal is a human. In some embodiments,the biological sample is a blood, serum, plasma, liver, amnioticmembrane, chorionic membrane or amniotic fluid sample. In someembodiments, the biological sample is a blood, serum, or plasma sample.In some embodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the HC1 of IαI is purified from human blood, plasma orserum. In some embodiments, IαI is isolated from human serum. In someembodiments, HC1 of IαI is not purified from serum. In some embodiments,HC1 of IαI is prepared by recombinant technology. In some embodiments,HC1 of IαI is purified from hepatic cells. In some embodiments, HC1 ofIαI is purified from amniotic membrane cells. In some embodiments, HC1of IαI is purified from amniotic epithelial cells or umbilical cordepithelial cells. In some embodiments, HC1 of IαI is purified fromamniotic stromal cells or umbilical cord stromal cells.

In some embodiments, HC1 comprises a polypeptide having the sequence setforth in SEQ ID NO: 47 or a polypeptide having at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 47.

In some embodiments, HC2 of IαI is isolated from a biological sample. Insome embodiments the biological sample is a biological sample from amammal. In some embodiments, the mammal is a human. In some embodiments,the biological sample is a blood, serum, plasma, liver, amnioticmembrane, chorionic membrane or amniotic fluid sample. In someembodiments, the biological sample is a blood, serum, or plasma sample.In some embodiments, the biological sample is a blood sample. In someembodiments, the biological sample is a serum sample. In someembodiments, the biological sample is a plasma sample. In someembodiments, the HC2 of IαI is purified from human blood, plasma orserum. In some embodiments, HC2 of IαI is isolated from human serum. Insome embodiments, HC2 of IαI is isolated from human serum. In someembodiments, HC2 of IαI is not isolated from blood serum. In someembodiments, HC2 of IαI is prepared by recombinant technology. In someembodiments, HC2 of IαI is purified from hepatic cells. In someembodiments, HC2 of IαI is purified from amniotic membrane cells. Insome embodiments, HC2 of IαI is purified from amniotic epithelial cellsor umbilical cord epithelial cells. In some embodiments, HC2 of IαI ispurified from amniotic stromal cells or umbilical cord stromal cells.

In some embodiments, HC2 comprises a polypeptide having the sequence setforth in SEQ ID NO: 49 or a polypeptide having at least 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity to thepolypeptide having the sequence set forth in SEQ ID NO: 49.

In some embodiments, IαI comprises bikunin. In some embodiments, bikunincomprises a polypeptide having the sequence set forth in SEQ ID NO: 53or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%,or 99% sequence amino acid identity to the polypeptide having thesequence set forth in SEQ ID NO: 53. In some embodiments, IαI comprisesa chondroitin sulfate chain.

HA

In some embodiments, HA is purified from a cell, tissue or a fluidsample. In some embodiments, HA is obtained from a commercial supplier(e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, CA (e.g.,Healon)). In some embodiments, HA is obtained from a commercial supplieras a powder. In some embodiments, HA is expressed in a cell. Exemplarycells suitable for the expression of HA include, but are not limited to,animal cells including, but not limited to, mammalian cells, primatecells, human cells, rodent cells, insect cells, bacteria, and yeast, andplant cells, including, but not limited to, algae, angiosperms,gymnosperms, pteridophytes and bryophytes. In some embodiments, HA isexpressed in a human cell. In some embodiments, HA is expressed in atransgenic animal. In some embodiments, HA is obtained from a cell thatexpresses a hyaluronan synthase (e.g., HAS1, HAS2, and HAS3). In someembodiments, the cell contains a recombinant expression vector thatexpresses an HA synthase. In certain instances, an HA synthase lengthenshyaluronan by repeatedly adding glucuronic acid and N-acetylglucosamineto the nascent polysaccharide as it is extruded through the cellmembrane into the extracellular space.

HA for use in the methods is typically high molecular weight (HMW) HA.In some embodiments, the weight average molecular weight of HMW HA isgreater than about 500 kilodaltons (kDa), such as, for example, betweenabout 500 kDa and about 10,000 kDa, between about 800 kDa and about8,500 kDa, between about 1100 kDa and about 5,000 kDa, or between about1400 kDa and about 3,500 kDa. In some embodiments, the weight averagemolecular weight of HMW HA is about 3000 kDa.

Additional Components

In some embodiments, one or more additional components are added togenerate an rcHC-HA/PTX3 complex. In some embodiments, a small leucinerich proteoglycan (SLRP) is added to generate an rcHC-HA/PTX3 complex.In some embodiments, the SLRP is a class I, class II or class II SLRP.In some embodiments, the SLRP is selected from among class I SLRPs, suchas decorin and biglycan. In some embodiments, the SLRP is selected fromamong class II SLRPs, such as fibromodulin, lumican, PRELP (prolinearginine rich end leucine-rich protein), keratocan, and osteoadherin. Insome embodiments, the SLRP is selected from among class III SLRPs, suchas epipycan and osteoglycin. In some embodiments, the SLRP is selectedfrom among bikunin, decorin, biglycan, and osteoadherin. In someembodiments, the SLRP comprises a glycosaminoglycan. In someembodiments, the SLRP comprises keratan sulfate.

HA Immobilization

In some embodiments, HMW HA is immobilized by any suitable method. Insome embodiments, HMW HA is immobilized to a solid support, such asculture dish, bead, a column or other suitable surfaces, such as, forexample, a surface of an implantable medical device or a portion thereofor on a surface that is subsequently connected to or combined with animplantable medical device as described herein. In some embodiments, HMWHA is immobilized directly to the solid support, such a by chemicallinkage. In some embodiments, HMW HA is attached indirectly to the solidsupport via a linker or an intermediary protein. Numerousheterobifunctional cross-linking reagents that are used to form covalentbonds between amino groups and thiol groups and to introduce thiolgroups into proteins, are known to those of skill in this art. In someembodiments, HMW HA is immobilized directly to the solid support viacrosslinking to the solid support. In some embodiments, HMW HA isimmobilized directly to the solid support without crosslinking to thesolid support. In some embodiments, HMW HA is immobilized directly tothe solid support as a coating. In some embodiments, HMW HA isimmobilized to a Covalink™-NH surface. In some embodiments, HMW HA isimmobilized directly to the solid support as a coating. In someembodiments, HMW HA is immobilized to a Covalink™-NH surface for about16 h at 4° C.

In some embodiments, the method comprises immobilizing HMW HA to a solidsurface via direct linkage to a solid support (i.e. without anintermediary protein). In some embodiments, the solid support is washedto remove unbound HMW HA prior to contacting the immobilized HA withPTX3. In some embodiments, the solid support is washed with washes of 8MGnHCl and PBS to remove unbound HMW HA prior to contacting theimmobilized HA with PTX3.

In some embodiments, the method comprises immobilizing HA to a solidsurface via an intermediary protein or a linker. In some embodiments,the linker is a peptide linker. In some embodiments, the intermediaryprotein is an HA binding protein (HABP). In some embodiments, HABP isfirst attached to a solid support (e.g., by cross-linking, chemicallinkage or via a chemical linker). In some embodiments, the solidsupport comprising HABP is then contacted with HA (e.g., HMW HA) toimmobilize HA to the solid support via binding of the HABP to HA. Insome embodiments, the solid support is washed to remove unbound HMW HAprior to contacting the immobilized HMW HA with PTX3. In someembodiments, the solid support is washed with washes of 8M GnHCl and PBSto remove unbound HMW HA prior to contacting the immobilized HA withPTX3.

In some embodiments, the method comprises immobilizing HA to a solidsurface via attachment of a peptide linker to the solid support andattachment HA to the peptide linker. In some embodiments, the peptidelinker comprises a protease cleavage site.

In some embodiments, the method comprises immobilizing HA to a solidsurface via attachment of a cleavable chemical linker, such as, but notlimited to a disulfide chemical linker.

In some embodiments, the HABP selected for use in the methods is an HABPthat is dissociated from HA following formation of the rcHC-HA/PTX3complex. In some embodiments, the HABP non-covalently binds to HA. Insome embodiments, the method further comprises dissociating thercHC-HA/PTX3 complex from HABP using one or more dissociating agents.Dissociating agents for the disruption of non covalent interactions(e.g., guanidine hydrochloride, urea and various detergents, e.g., SDS)are known in the art. In some embodiments the dissociating agent isurea. In some embodiments the dissociating agent is guanidinehydrochloride. In some embodiments, the dissociation agent is about 4Mto about 8M guanidine-HCl. In some embodiments, the dissociation agentis about 4M, about 5M, about 6M, about 7M, about 8M guanidine-HCl. Insome embodiments, the dissociation agent is about 4M to about 8Mguanidine-HCl in PBS at pH 7.5.

In some embodiments, such dissociating agents are employed to dissociatethe rcHC-HA/PTX3 complex from an intermediary HABP. An HABP for use inthe methods typically is selected such that the binding affinity for HAis strong enough to permit assembly of the rcHC-HA/PTX3 complex but isdissociated from the rcHC-HA/PTX3 complex with a suitable dissociationagent. In some embodiments the dissociating agent is guanidinehydrochloride.

Exemplary HABPs for use with the methods provided herein include, butare not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggrecan, versican,neurocan, brevican, phosphacan, TSG-6, CD44, stabilin-1, stabilin-2, orportions thereof (e.g., link modules thereof) sufficient to bind HA. Insome embodiments, the HABP comprises a polypeptide having the sequenceset forth in any of SEQ ID NOS: 54-99 or a polypeptide having at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acididentity to the polypeptide having the sequence set forth in any of SEQID NOS: 54-99. In some embodiments, the HABP is versican. In someembodiments, the HABP is a recombinant protein. In some embodiments, theHABP is a recombinant mammalian protein. In some embodiments, the HABPis a recombinant human protein. In some embodiments, the HABP is arecombinant versican protein or a portion thereof sufficient to bind toHA. In some embodiments, the HABP is a recombinant aggrecan protein or aportion thereof sufficient to bind to HA. In some embodiments, the HABPis a native HABP or a portion thereof sufficient to bind to HA. In someembodiments, the native HABP is isolated from mammalian tissue or cells.In some embodiments, the HABP is isolated from bovine nasal cartilage(e.g. HABP from Seikagaku which contains the HA binding domains ofaggrecan and link protein).

In some embodiments, the HABP comprises a link module of HAPLN1, HAPLN2,HAPLN3, HAPLN4, aggrecan, versican, neurocan, brevican, phosphacan,TSG-6, CD44, stabilin-1, or stabilin-2. In some embodiments, the HABPcomprising a link module comprises a polypeptide having the sequence setforth in any of link domains of SEQ ID NOS: 54-99 or a polypeptidehaving at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequenceamino acid identity to the polypeptide having the sequence set forth inany of link domains of SEQ ID NOS: 54-99. In some embodiments, the HABPcomprises a link module of versican. In some embodiments, the HABPcomprising a link module is a recombinant protein. In some embodiments,the HABP comprising a link module of versican is a recombinant protein.

In some embodiments, the or intermediary protein, such as an HABP,contains a proteolytic cleavage sequence that is recognized by and ishydrolyzed by a site specific protease, such as furin, 3C protease,caspase, matrix metalloproteinase or TEV protease. In such embodiments,assembled rcHC-HA/PTX3 complexes are released from the solid support bycontacting the immobilized complexes with a protease that cleaves thespecific cleavage sequence.

In some embodiments, the rcHC-HA/PTX3 complex is purified. In someembodiments, the rcHC-HA/PTX3 complex is purified by any suitable methodor combination of methods. The embodiments described below are notintended to be exclusive, only exemplary.

In some embodiments, the rcHC-HA/PTX3 complex is purified bychromatography (e.g., ion exchange, affinity, size exclusion, andhydroxyapatite chromatography), gel filtration, centrifugation (e.g.,gradient centrifugation), or differential solubility, ethanolprecipitation or by any other available technique for the purificationof proteins.

In some embodiments, the rcHC-HA/PTX3 complex is purified byimmunoaffinity chromatography. In some embodiments antibodies aregenerated against a component of the rcHC-HA/PTX3 complex (e.g.,anti-HC1, anti-PTX, an antibody against one or more SLRPs of thercHC-HA/PTX3 complex, e.g., anti-bikunin, anti-decorin, anti-biglycan,or anti-osteoadherin) and affixed to a solid support. In someembodiments, the unpurified rcHC-HA/PTX3 complex (i.e., the mobilephase) is passed over the support. In certain instances, thercHC-HA/PTX3 complex binds to the antibodies. In some embodiments, thesupport is washed (e.g., with PBS) to remove any unbound or looselybound molecules. In some embodiments, the support is then washed with asolution that enables elution of the rcHC-HA/PTX3 complex from thesupport (e.g., 1% SDS, 6 M guanidine-HCl, or 8 M urea). In someembodiments, the dissociating agent is removed from the dissociatedrcHC-HA/PTX3 complex. In some embodiments, the dissociating agent isremoved from the dissociated rcHC-HA/PTX3 complex by a method including,but not limited to, ion-exchange chromatography, dialysis, gelfiltration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by affinitychromatography. In some embodiments, an HABP is employed to bind to thercHC-HA/PTX3 complex for purification of the complex and affixed to astationary support. In some embodiments, the unpurified rcHC-HA/PTX3complex (i.e., the mobile phase) is passed over the support. In certaininstances, the rcHC-HA/PTX3 complex binds to the HABP. In someembodiments the support is washed (e.g., with PBS) to remove any unboundor loosely bound molecules. In some embodiments, the support is thenwashed with a solution (e.g., a dissociating agent) that enables elutionof the rcHC-HA/PTX3 complex from the support. In some embodiments, thedissociating agent is removed from the dissociated rcHC-HA/PTX3 complexby a method including, but not limited to, ion-exchange chromatography,dialysis, gel filtration chromatography, ultrafiltration, ordiafiltration.

In some embodiments, the rcHC-HA/PTX3 complex is purified by acombination of HABP affinity chromatography, and immunoaffinitychromatography using antibodies against one or more components of thercHC-HA/PTX3 complex.

In some embodiments, one or more components of the rcHC-HA/PTX3 complexdisclosed herein comprise an affinity tag (e.g., a fusion protein ofPTX3 or HC1 with an affinity tag). Exemplary affinity tags that areincorporated into one or more components of the rcHC-HA/PTX3 complex insome embodiments include, but are not limited to, a hemagglutinin tag,poly-histidine, a myc tag, a FLAG tag, or glutathione-S-transferasesequence. In some embodiments, the ligand for the affinity tag isaffixed to the solid support. In some embodiments, the unpurifiedrcHC-HA/PTX3 complex is passed over the support. In certain instances,the rcHC-HA/PTX3 complex binds to the ligand. In some embodiments thesupport is washed (e.g., with PBS) to remove any unbound or looselybound molecules. In some embodiments, the support is then washed with asolution that enables elution of an rcHC-HA/PTX3 complex disclosedherein from the support. In some embodiments, the elution agent isremoved from the dissociated rcHC-HA/PTX3 complex by a method including,but not limited to, ion-exchange chromatography, dialysis, gelfiltration chromatography, ultrafiltration, or diafiltration.

In some embodiments, the PTX3, TSG-6, and/or HC1 are conjugated to alabel. A “label” refers to a detectable compound or composition which isconjugated directly or indirectly to a polypeptide so as to generate alabeled polypeptide. In some embodiments, the label is detectable byitself (e.g., radioisotope labels or fluorescent labels) or, in the caseof an enzymatic label, catalyzes chemical alteration of a substratecompound composition which is detectable. Non-limiting examples oflabels include fluorogenic moieties, dyes, fluorescent tags, greenfluorescent protein, or luciferase.

Methods of Assessing the Activity of nHC-HA/PTX3 and rcHC-HA/PTX3Complexes

The properties of nHC-HA/PTX3 and rcHC-HA/PTX3 complexes provided hereinare assessed by any suitable method including, in vitro and in vivomethods. Exemplary in vitro methods are provided herein and include, butare not limited, to cell culture methods that assess the ability ofnHC-HA/PTX3 or rcHC-HA/PTX3 complexes to promote attachment ofmacrophages to the immobilized nHC-HA/PTX3 or rcHC-HA/PTX3 complexes, toinhibit or reduce aggregation of macrophages, to promote apoptosis ofneutrophils, macrophage phagocytosis of apoptotic neutrophils, and M2polarization of stimulated macrophages. In some embodiments, themacrophages used in the assay are stimulated, such as by exposure to LPSor IFN-y. In some embodiments, the gene or protein expression instimulated macrophages is assessed following contact with nHC-HA/PTX3 orrcHC-HA/PTX3 complexes. In such methods of assessing activity ofnHC-HA/PTX3 or rcHC-HA/PTX3 complex, a suitable control is employed forcomparison. In some embodiments, the control is the absence of treatmentwith an nHC-HA/PTX3 or rcHC-HA/PTX3 complex (i.e. a negative control).

In some embodiments, the activity of an rcHC-HA/PTX3 complex is comparedto the activity of a native HC-HA/PTX3 complex. In some embodiments thenative HC-HA/PTX3 is isolated from amniotic membrane.

In some embodiments, gene expression in treated macrophages is assessedby PCR, RT-PCR, Northern blotting, western blotting, dot blotting,immunohistochemistry, chromatography or other suitable method ofdetecting proteins or nucleic acids. In some embodiments, the level ofexpression of IL-10, IL-12, IL23, LIGHT and SPHK1 is assessed.

Exemplary in vitro methods for assessing the activity of an nHC-HA/PTX3or rcHC-HA/PTX3 complex provided herein include, but are not limited, toanimal models of various disease and conditions. A variety of animalmodels are available and well-known in the art for diseases andconditions, including, but not limited to, animal models (e.g. rodentand primate models) for various inflammatory and autoimmune diseases anddisorders including, but not limited to, ischemia reperfusion injury,type 1 and type 2 diabetes, inflammatory diseases, collagen inducedarthritis, rheumatoid arthritis, antigen induced autoimmune disease suchas collagen induced arthritis and myelin peptide-induced experimentalallergic encephalomyelitis, inflammatory bowel disease (IBD)/ulcerativecolitis, multiple sclerosis, surgically induced osteoarthritis andnephritis, psoriasis, inflammatory skin diseases, LPS-induced endotoxicshock, LPS-induced lung injury, allergic rhinitis, liver injury, chronicstress, asthma, and xenograft and allograft models for various cancers.

In some embodiments, the animal model is a rodent model of inflammationsuch as chronic graft-versus-host disease (cGVHD), HSV1-inducednecrotizing stromal keratitis, or high-risk corneal transplantation. Insome embodiments, reduction of inflammation by nHC-HA/PTX3 orrcHC-HA/PTX3 treatment is assessed by the measuring the proliferationand activation of T cells and the production of immune cytokines such asIL-1α, IL-2, IL-6, IFN-y, and TNF-α. In some embodiments, the animalmodel is a rodent model of scarring such as excimer laser-assistedphotorefractive keratectomy (PRK). Exemplary methods for the use of suchanimal models are provided in the Examples provided herein.

In some embodiments, the animal model is a genetic model of inflammatoryand autoimmune diseases and disorder that contains one or more geneticmodifications that cause the disease or disorder. In some embodiments,such models are obtained from a commercial source. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein is administeredto an animal model of a particular disease or condition and the abilityof the rcHC-HA/PTX3 complex to inhibit or reduce one or more symptoms ofthe disease or condition is assessed.

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complexes describedherein. Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complexes producedby the methods provided herein. In some embodiments, the pharmaceuticalcompositions are formulated in a conventional manner using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of an nHC-HA/PTX3 or rcHC-HA/PTX3 complexinto preparations which are suitable for pharmaceutical use. Properformulation is dependent upon the route of administration selected. Anyof the well-known techniques, carriers, and excipients can be used assuitable and as understood in the art.

Disclosed herein, in certain embodiments, is a pharmaceuticalcomposition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein. In some embodiments, the pharmaceutical composition furthercomprises at least one pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition further comprises anadjuvant, excipient, preservative, agent for delaying absorption,filler, binder, adsorbent, buffer, and/or solubilizing agent. Exemplarypharmaceutical compositions that are formulated to contain annHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein include, but are notlimited to, a solution, suspension, emulsion, syrup, granule, powder,ointment, tablet, capsule, pill or an aerosol.

Dosage Forms

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered as an aqueous suspension. In some embodiments, anaqueous suspension comprises water, Ringer’s solution and/or isotonicsodium chloride solution. In some embodiments, an aqueous suspensioncomprises a sweetening or flavoring agent, coloring matters or dyes and,if desired, emulsifying agents or suspending agents, together withdiluents water, ethanol, propylene glycol, glycerin, or combinationsthereof. In some embodiments, an aqueous suspension comprises asuspending agent. In some embodiments, an aqueous suspension comprisessodium carboxymethylcellulose, methylcellulose,hydroxypropylmethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone,gum tragacanth and/or gum acacia. In some embodiments, an aqueoussuspension comprises a dispersing or wetting agent. In some embodiments,an aqueous suspension comprises a naturally-occurring phosphatide, forexample lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethylene-oxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate. Insome embodiments, an aqueous suspension comprises a preservative. Insome embodiments, an aqueous suspension comprises ethyl, or n-propylp-hydroxybenzoate. In some embodiments, an aqueous suspension comprisesa sweetening agent. In some embodiments, an aqueous suspension comprisessucrose, saccharin or aspartame.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered as an oily suspension. In some embodiments, anoily suspension is formulated by suspending the active ingredient in avegetable oil (e.g., arachis oil, olive oil, sesame oil or coconut oil),or in mineral oil (e.g., liquid paraffin). In some embodiments, an oilysuspension comprises a thickening agent (e.g., beeswax, hard paraffin orcetyl alcohol). In some embodiments, an oily suspension comprisessweetening agents (e.g., those set forth above). In some embodiments, anoily suspension comprises an anti-oxidant (e.g., butylated hydroxyanisolor alpha-tocopherol).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated for parenteral injection (e.g., via injection orinfusion, including intraarterial, intracardiac, intradermal,intraduodenal, intramedullary, intramuscular, intraosseous,intraperitoneal, intrathecal, intravascular, intravenous, intravitreal,epidural and/or subcutaneous). In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered as a sterilesolution, suspension or emulsion.

In some embodiments, a formulation for parenteral administrationincludes aqueous and/or non-aqueous (oily) sterile injection solutionsof an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein, which insome embodiments, contain antioxidants, buffers, bacteriostats and/orsolutes which render the formulation isotonic with the blood of theintended recipient; and/or aqueous and/or non-aqueous sterilesuspensions which in some embodiments, include a suspending agent and/ora thickening agent. In some embodiments, a formulation for parenteraladministration includes suitable stabilizers or agents which increasethe solubility of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein to allow for the preparation of highly concentrated solutions.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered as an oil-in-water micro-emulsion where theactive ingredient is dissolved in the oily phase. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved ina fatty oil (e.g., sesame oil, or synthetic fatty acid esters, (e.g.,ethyl oleate or triglycerides, or liposomes. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is dissolved in amixture of soybean oil and/or lecithin. In some embodiments, the oilsolution is introduced into a water and glycerol mixture and processedto form a micro-emulsion.

In some embodiments, a composition formulated for parenteraladministration is administered as a single bolus shot. In someembodiments, a composition formulated for parenteral administration isadministered via a continuous intravenous delivery device (e.g., DeltecCADD-PLUS™ model 5400 intravenous pump).

In some embodiments, a formulation for injection is presented in unitdosage form, e.g., in ampoules or in multi-dose containers, with anadded preservative. In some embodiments, a formulation for injection isstored in powder form or in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for example,saline or sterile pyrogen-free water, immediately prior to use.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated for topical administration. Topical formulationsinclude, but are not limited to, ointments, creams, lotions, solutions,pastes, gels, films, sticks, liposomes, nanoparticles. In someembodiments, a topical formulation is administered by use of a patch,bandage or wound dressing.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated as composition is in the form of a solid, across-linked gel, or a liposome. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is formulated as an insolublecross-linked hydrogel.

In some embodiments, a topical formulation comprises a gelling (orthickening) agent. Suitable gelling agents include, but are not limitedto, celluloses, cellulose derivatives, cellulose ethers (e.g.,carboxymethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxymethylcellulose, hydroxypropylmethylcellulose,hydroxypropylcellulose, methylcellulose), guar gum, xanthan gum, locustbean gum, alginates (e.g., alginic acid), silicates, starch, tragacanth,carboxyvinyl polymers, carrageenan, paraffin, petrolatum, acacia (gumarabic), agar, aluminum magnesium silicate, sodium alginate, sodiumstearate, bladderwrack, bentonite, carbomer, carrageenan, carbopol,xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia,chondrus, dextrose, furcellaran, gelatin, ghatti gum, guar gum,hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey,maize starch, wheat starch, rice starch, potato starch, gelatin,sterculia gum, polyethylene glycol (e.g. PEG 200-4500), gum tragacanth,ethyl cellulose, ethylhydroxyethyl cellulose, ethylmethyl cellulose,methyl cellulose, hydroxyethyl cellulose, hydroxyethylmethyl cellulose,hydroxypropyl cellulose, poly(hydroxyethyl methacrylate),oxypolygelatin, pectin, polygeline, povidone, propylene carbonate,methyl vinyl ether/maleic anhydride copolymer (PVM/MA),poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate),hydroxypropyl cellulose, hydroxypropylmethyl-cellulose (HPMC), sodiumcarboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone(PVP: povidone), or combinations thereof.

In some embodiments, a topical formulation disclosed herein comprises anemollient. Emollients include, but are not limited to, castor oilesters, cocoa butter esters, safflower oil esters, cottonseed oilesters, corn oil esters, olive oil esters, cod liver oil esters, almondoil esters, avocado oil esters, palm oil esters, sesame oil esters,squalene esters, kikui oil esters, soybean oil esters, acetylatedmonoglycerides, ethoxylated glyceryl monostearate, hexyl laurate,isohexyl laurate, isohexyl palmitate, isopropyl palmitate, methylpalmitate, decyloleate, isodecyl oleate, hexadecyl stearate decylstearate, isopropyl isostearate, methyl isostearate, diisopropyladipate, diisohexyl adipate, dihexyldecyl adipate, diisopropyl sebacate,lauryl lactate, myristyl lactate, and cetyl lactate, oleyl myristate,oleyl stearate, and oleyl oleate, pelargonic acid, lauric acid, myristicacid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid,oleic acid, linoleic acid, ricinoleic acid, arachidic acid, behenicacid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol,hexadecyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearylalcohol, oleyl alcohol, ricinoleyl alcohol, behenyl alcohol, erucylalcohol, 2-octyl dodecanyl alcohol, lanolin and lanolin derivatives,beeswax, spermaceti, myristyl myristate, stearyl stearate, carnauba wax,candelilla wax, lecithin, and cholesterol.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated with one or more natural polymers. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isformulated with a natural polymer that is fibronectin, collagen,laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate,chondroitin sulfate. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is formulated with a polymer gel formulatedfrom a natural polymer. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is formulated with a polymer gelformulated from a natural polymer, such as, but not limited to,fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronicacid, heparan sulfate, chondroitin sulfate, and combinations thereof. Insome embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated with a cross-linked polymer. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulatedwith a non-cross-linked polymer. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is formulated with anon-cross-linked polymer and a cross-linked polymer. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isformulated with cross-linked hyaluronan gel. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated withan insoluble cross-linked HA hydrogel. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is formulated withnon-cross-linked hyaluronan gel. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is formulated with a collagenmatrix. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is formulated with a fibrin matrix. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isformulated with a fibrin/collagen matrix.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated for administration to an eye or a tissue relatedthereto. Formulations suitable for administration to an eye include, butare not limited to, solutions, suspensions (e.g., an aqueoussuspension), ointments, gels, creams, liposomes, niosomes,pharmacosomes, nanoparticles, or combinations thereof. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein fortopical administration to an eye is administered spraying, washing, orcombinations thereof. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered to an eye via aninjectable depot preparation.

As used herein, a “depot preparation” is a controlled-releaseformulation that is implanted in an eye or a tissue related thereto(e.g., the sclera) (for example subcutaneously, intramuscularly,intravitreally, or within the subconjunctiva). In some embodiments, adepot preparation is formulated by forming microencapsulated matrices(also known as microencapsulated matrices) of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein in biodegradable polymers. In someembodiments, a depot preparation is formulated by entrapping annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein in liposomes ormicroemulsions.

A formulation for administration to an eye has an ophthalmicallyacceptable tonicity. In certain instances, lacrimal fluid has anisotonicity value equivalent to that of a 0.9% sodium chloride solution.In some embodiments, an isotonicity value from about 0.6% to about 1.8%sodium chloride equivalency is suitable for topical administration to aneye. In some embodiments, a formulation for administration to an eyedisclosed herein has an osmolarity from about 200 to about 600 mOsm/L.In some embodiments, a formulation for administration to an eyedisclosed herein is hypotonic and thus requires the addition of anysuitable to attain the proper tonicity range. Ophthalmically acceptablesubstances that modulate tonicity include, but are not limited to,sodium chloride, potassium chloride, sodium thiosulfate, sodiumbisulfite and ammonium sulfate.

A formulation for administration to an eye has an ophthalmicallyacceptable clarity. Examples of ophthalmically-acceptable clarifyingagents include, but are not limited to, polysorbate 20, polysorbate 80,or combinations thereof.

In some embodiments, a formulation for administration to an eyecomprises an ophthalmically acceptable viscosity enhancer. In someembodiments, a viscosity enhancer increases the time a formulationdisclosed herein remains in an eye. In some embodiments, increasing thetime a formulation disclosed herein remains in the eye allows forgreater drug absorption and effect. Non-limiting examples ofmucoadhesive polymers include carboxymethylcellulose, carbomer (acrylicacid polymer), poly(methylmethacrylate), polyacrylamide, polycarbophil,acrylic acid/butyl acrylate copolymer, sodium alginate and dextran.

In some embodiments, a formulation for administration to an eye isadministered or delivered to the posterior segments of an eye (e.g., tothe retina, choroid, vitreous and optic nerve). In some embodiments, atopical formulation for administration to an eye disclosed herein fordelivery to the posterior of the eye comprises a solubilizing agent, forexample, a glucan sulfate and/or a cyclodextrin. Glucan sulfates whichare used in some embodiments include, but are not limited to, dextransulfate, cyclodextrin sulfate and β-1,3-glucan sulfate, both natural andderivatives thereof, or any compound which temporarily binds to and beretained at tissues which contain fibroblast growth factor (FGF), whichimproves the stability and/or solubility of a drug, and/or whichimproves penetration and ophthalmic absorption of a topical formulationfor administration to an eye disclosed herein. Cyclodextrin derivativeswhich are used in some embodiments as a solubilizing agent include, butare not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin,hydroxyethyl β -cyclodextrin, hydroxypropyl γ -cyclodextrin,hydroxypropyl β-cyclodextrin, sulfated α -cyclodextrin, sulfated β-cyclodextrin, sulfobutyl ether β -cyclodextrin.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is formulated for rectal or vaginal administration. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered as a suppository. In some embodiments, a compositionsuitable for rectal administration is prepared by mixing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will therefore melt in the rectum to release thedrug. In some embodiments, a composition suitable for rectaladministration is prepared by mixing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein with cocoa butter, glycerinated gelatin,hydrogenated vegetable oils, mixtures of polyethylene glycols of variousmolecular weights or fatty acid esters of polyethylene glycol.

In certain embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex describedherein is optionally incorporated within controlled release particles,lipid complexes, liposomes, nanoparticles, microspheres, microparticles,nanocapsules or other agents which enhance or facilitate localizeddelivery to the skin. An example of a conventional microencapsulationprocess for pharmaceutical preparations is described in U.S. Pat. No.3,737,337, incorporated herein by reference for such disclosure.

Dosages

The amount of pharmaceutical compositions administered is dependent inpart on the individual being treated. In instances where pharmaceuticalcompositions are administered to a human subject, the daily dosage willnormally be determined by the prescribing physician with the dosagegenerally varying according to the age, sex, diet, weight, generalhealth and response of the individual, the severity of the individual’ssymptoms, the precise disease or condition being treated, the severityof the disease or condition being treated, time of administration, routeof administration, the disposition of the composition, rate ofexcretion, drug combination, and the discretion of the prescribingphysician.

In some embodiments, the dosage of an nHC-HA/PTX3 or rcHC-HA/PTX3complex is between about 0.001 to about 1000 mg/kg body weight/day. Insome embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is in the range of about 0.5 to about 50 mg/kg/day. Insome embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is about 0.001 to about 7 g/day. In some embodiments,the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isabout 0.01 to about 7 g/day. In some embodiments, the amount ofnHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is about 0.02 toabout 5 g/day. In some embodiments, the amount of nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is about 0.05 to about 2.5 g/day.In some embodiments, the amount of nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is about 0.1 to about 1 g/day.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered, before, during or after the occurrence of adisease or condition. In some embodiments, a combination therapy isadministered before, during or after the occurrence of a disease orcondition. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is administered with a combination therapy before,during or after the occurrence of a disease or condition. In someembodiments, the timing of administering the composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 disclosed herein varies. Thus, in someexamples, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused as a prophylactic and is administered continuously to subjects witha propensity to develop conditions or diseases in order to prevent theoccurrence of the disease or condition. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is administered toa subject during or as soon as possible after the onset of the symptoms.In some embodiments, the administration of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is initiated within the first 48hours of the onset of the symptoms, preferably within the first 48 hoursof the onset of the symptoms, more preferably within the first 6 hoursof the onset of the symptoms, and most preferably within 3 hours of theonset of the symptoms. In some embodiments, the initial administrationis via any route practical, such as, for example, an intravenousinjection, a bolus injection, infusion over 5 minutes to about 5 hours,a pill, a capsule, transdermal patch, buccal delivery, or combinationthereof. An nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein ispreferably administered as soon as is practicable after the onset of adisease or condition is detected or suspected, and for a length of timenecessary for the treatment of the disease, such as, for example, fromabout 1 month to about 3 months. In some embodiments, the length oftreatment varies for each subject, and the length is determined usingthe known criteria. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein or a formulation containing a complex isadministered for at least 2 weeks, preferably about 1 month to about 5years, and more preferably from about 1 month to about 3 years.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered in a single dose, once daily. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered in multiple doses, more than once per day. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered twice daily. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered three times perday. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex isadministered four times per day. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered more than fourtimes per day.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered for prophylactic and/or therapeutic treatments.In therapeutic applications, in some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered to an individualalready suffering from a disease or condition, in an amount sufficientto cure or at least partially arrest the symptoms of the disease orcondition. Amounts effective for this use will depend on the severityand course of the disease or condition, previous therapy, theindividual’s health status, weight, and response to the drugs, and thejudgment of the treating physician.

In prophylactic applications, in some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered to an individualthat is at risk of a particular disorder. Such an amount is defined tobe a “prophylactically effective amount or dose.” In such use, theprecise amounts also depend on the individual’s state of health, weight,and other physical parameters of the individual.

In the case wherein the individual’s condition does not improve, uponthe doctor’s discretion an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered chronically, that is, for an extended period oftime, including throughout the duration of the individual’s life inorder to ameliorate or otherwise control or limit the symptoms of theindividual’s disease or condition.

In some embodiments, in cases where the individual’s status doesimprove, upon the doctor’s discretion, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is administered continuously or the dose ofdrug being administered is temporarily reduced or temporarily suspendedfor a certain length of time (i.e., a “drug holiday”). In someembodiments, the length of the drug holiday varies between 2 days and 1year, including by way of example only, 2 days, 3 days, 4 days, 5 days,6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250days, 280 days, 300 days, 320 days, 350 days, or 365 days. In someembodiments the dose reduction during a drug holiday is from 10%-100%,including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the individual’s conditions has occurred, amaintenance dose is administered if necessary. In some embodiments,subsequently, the dosage or the frequency of administration, or both, isreduced, as a function of the symptoms, to a level at which the improveddisease, disorder or condition is retained. In some embodiments,individuals require intermittent treatment on a long-term basis upon anyrecurrence of symptoms.

In some embodiments, the pharmaceutical composition described herein isin unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein. In some embodiments, the unit dosage is in theform of a package containing discrete quantities of the formulation.Non-limiting examples are packaged tablets or capsules, and powders invials or ampoules. In some embodiments, aqueous suspension compositionsare packaged in single-dose non-reclosable containers. In someembodiments, multiple-dose reclosable containers are used, in which caseit is typical to include a preservative in the composition. In someembodiments, formulations for parenteral injection are presented in unitdosage form, which include, but are not limited to ampoules, or in multidose containers, with an added preservative.

The daily dosages appropriate for an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein are, for example, from about 0.01 to 2.5 mg/kg per bodyweight. An indicated daily dosage in the larger mammal, including, butnot limited to, humans, is in the range from about 0.5 mg to about 100mg, conveniently administered in divided doses, including, but notlimited to, up to four times a day or in extended release form. Suitableunit dosage forms for oral administration include from about 1 to 50 mgactive ingredient. The foregoing ranges are merely suggestive, as thenumber of variables in regard to an individual treatment regime islarge, and considerable excursions from these recommended values are notuncommon. In some embodiments, the dosages are altered depending on anumber of variables, not limited to the activity of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex used, the disease or condition to be treated, themode of administration, the requirements of the individual subject, theseverity of the disease or condition being treated, and the judgment ofthe practitioner.

In some embodiments, the toxicity and therapeutic efficacy of suchtherapeutic regimens are determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, including, but notlimited to, the determination of the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). In some embodiments, the dose ratio between the toxicand therapeutic effects is the therapeutic index and it is expressed asthe ratio between LD₅₀ and ED₅₀. nHC-HA/PTX3 or rcHC-HA/PTX3 complexesexhibiting high therapeutic indices are preferred. In some embodiments,the data obtained from cell culture assays and animal studies is used informulating a range of dosage for use in human. The dosage of annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein lies preferablywithin a range of circulating concentrations that include the ED₅₀ withminimal toxicity. In some embodiments, the dosage varies within thisrange depending upon the dosage form employed and the route ofadministration utilized.

In some embodiments, the pharmaceutical compositions of nHC-HA/PTX3 orrcHC-HA/PTX3 complexes are packaged as articles of manufacturecontaining packaging material, a pharmaceutical composition which iseffective for prophylaxis and/or treating a disease or condition, and alabel that indicates that the pharmaceutical composition is to be usedfor treating the disease or condition. In some embodiments, thepharmaceutical compositions are packaged in unit dosage forms contain anamount of the pharmaceutical composition for a single dose or multipledoses. In some embodiments, the packaged compositions contain alyophilized powder of the pharmaceutical compositions, which isreconstituted (e.g., with water or saline) prior to administration.

Medical Device and Biomaterials Compositions

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is assembled directly on a surface of or formulated as a coatingfor an implantable medical device. Methods for covalent attachment ofhyaluronan to surfaces such as, but not limited to, metallic, polymeric,ceramic, silica and composite surfaces is well-known in the art and insome embodiments, is employed in conjunction with the methods providedherein for the assembly of nHC-HA/PTX3 or rcHC-HA/PTX3 complexes on suchsurfaces (see e.g., U.S. Pat. Nos. 5,356,433; 5,336,518, 4,613,665,4,810,784, 5,037,677, 8,093,365). In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex is assembled directly on a surface of animplantable medical device or a portion thereof. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex that has been generated accordingthe methods provided herein is purified and then attached directly on asurface of an implantable medical device or a portion thereof. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex that has beengenerated according the methods provided herein is purified and thenformulated as a coating for attachment to the medical device or aportion thereof. In some embodiments, the coating is applied directly tothe surfaces or is applied to a pretreated or coated surface where thepretreatment or coating is designed to aid adhesion of the coating tothe substrate. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex that has been generated according the methods provided herein ispurified and then attached to a medical device or a portion thereof thathas been coated with a substance that promotes the attachment of thenHC-HA/PTX3 or rcHC-HA/PTX3 complex. For example, in some embodiments,the medical device or a portion thereof is coated with an adhesivepolymer that provides functional groups on its surface for the covalentattachment of hyaluronan of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. Insome embodiments, a coupling agent, such as, but not limited tocarbodiimide is employed to attach the nHC-HA/PTX3 or rcHC-HA/PTX3complex to the polymer coating. In some embodiments, photoimmobilizationis employed to covalently attach an nHC-HA/PTX3 or rcHC-HA/PTX3 complexthat has been generated according the methods provided herein to medicaldevice or a portion thereof. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex that has been generated according the methodsprovided herein is attached to a medical device or a portion thereofusing a spacer molecule that comprises a photochemically orthermochemically reactive group.

In some embodiments, the coating formulations comprising an nHC-HA/PTX3or rcHC-HA/PTX3 complex are applied to the substrate by for exampledip-coating. Other methods of application include, but are not limitedto, spray, wash, vapor deposition, brush, roller, curtain, spin coatingand other methods known in the art.

Exemplary implantable medical devices include, but are not limited to anartificial joint, orthopedic device, bone implant, contact lenses,suture, surgical staple, surgical clip, catheter, angioplasty balloon,sensor, surgical instrument, electrode, needle, syringe, wound drain,shunt, urethral insert, metal or plastic implant, heart valve,artificial organ, lap band, annuloplasty ring, guide wire, K-wire orDenham pin, stent, stent graft, vascular graft, pacemaker, pellets,wafers, medical tubing, infusion sleeve, implantable defibrillator,neurostimulator, glucose sensor, cerebrospinal fluid shunt, implantabledrug pump, spinal cage, artificial disc, ocular implant, cochlearimplant, breast implant, replacement device for nucleus pulposus, eartube, intraocular lens, drug delivery system, microparticle,nanoparticle, and microcapsule.

In particular embodiments, the implantable medical device is an implantor prosthesis comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein. In particular embodiments, the prosthesis is anartificial joint. In some embodiments, the prosthesis is an artificialhip joint, artificial knee, an artificial glenohumeral joint, anartificial ankle.

In particular embodiments, the implant is a stent. In particularembodiments, the implant is a coronary stent, a ureteral stent, aurethral stent, a prostatic stent, a bone stent, or an esophageal stent.In particular embodiments, the implant is a bone implant, such as, butnot limited to, an osseointegrated implant or a craniofacial prosthesis(e.g., an artificial ear, orbital prosthesis, nose prosthesis).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is assembled directly on a microparticle or a nanoparticle fordelivery of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex to a subject (see,e.g., WO 03/015755 and US2004/0241248).

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes providedherein are attached to, assembled on, or provided as a coating on thesurfaces of or portions thereof of any such implantable medical devicesas described herein or known in the art. In some embodiments thenHC-HA/PTX3 or rcHC-HA/PTX3 complex elutes from the coating and into thesurrounding tissue following implantation.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is assembled directly on a scaffold, a microparticle, amicrocapsule or microcarrier employed for the delivery of a biomaterial,such as a stem cell or an insulin producing cell. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is attached tothe microcapsule or assembled directly on a microcapsule. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is combined with amaterial used to form the microcapsule and a microcapsule is generatedthat contains the nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is used to coat theinner surface of the microcapsule. In some embodiments, the nHC-HA/PTX3or rcHC-HA/PTX3 complex is used to coat the outer surface of themicrocapsule. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complex is used to coat the inner and outer surface of the microcapsule.

Exemplary materials for encapsulating cells include, but are not limitedto, thermosetting hydrogels, such as agarose, alginate, and artificialpolymers such as poly(NiPAAm-co-AAC), poly(ethylene glycol) (PEG) andPEG derivatives such as PEG diacrylate and oligo(poly(ethylene glycol))fumerate. Methods for the culturing and microencapsulation of stem cellsare known in the art in some embodiments, are employed to generatemicrocapsules containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex providedherein.

In some embodiments the microcapsule contains a cell, a plurality ofcells or other biological material. In some embodiments, the cell orcells are stem cells, such as, but not limited to, mesenchymal stemcells. In some embodiments, the cell or cells are differentiated cells,such as, but not limited to, insulin-producing cells. In someembodiments, the cell or cells are autologous cells (i.e. cells that arefrom or derived from the recipient of the cells). In some embodiments,the cell or cells are allogeneic cells (i.e. cells that are not from orderived from the recipient of the cells). In some embodiments, themicrocapsule contains a cell, a plurality of cells or other biologicalmaterial and the inner surfaces of the microcapsule are coated withnHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein. In some embodimentsthe microcapsule contains a cell, a plurality of cells or otherbiological material and the outer surfaces of the microcapsule arecoated with nHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein. In someembodiments the microcapsule contains a cell, a plurality of cells orother biological material and the outer and inner surfaces of themicrocapsule are coated with nHC-HA/PTX3 or rcHC-HA/PTX3 complexprovided herein. In some embodiments the microcapsule is administered totreat a disease or condition. Exemplary diseases and conditions andmethods of treatment for which a microcapsule can be administered aredescribed elsewhere herein and include but are not limited toinflammatory and immune related diseases.

Methods of Treatment

Disclosed herein, in certain embodiments, are methods of treating anindividual in need thereof, comprising administering to the individualnHC-HA/PTX3 or rcHC-HA/PTX3 complexes described herein. Disclosedherein, in certain embodiments, are methods of treating an individual inneed thereof, comprising administering to the individual nHC-HA/PTX3 orrcHC-HA/PTX3 complexes produced by the methods described herein. Thefollowing are non-limiting examples of methods of treatment comprisingadministration of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is used to inhibit at least one of the following:scarring, inflammation, immune reaction leading to autoimmune or immunerejection, adhesion, angiogenesis and conditions requiring cell ortissue regeneration. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is used to promote wound healing. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused to promote stem cell expansion. In some embodiments, an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is used to promote tissueregeneration.

In some embodiments, the methods of treating an individual in needthereof, comprising administering to the individual nHC-HA/PTX3 orrcHC-HA/PTX3 complexes described herein by any suitable method. In someembodiments, the methods of treating an individual in need thereof,comprising administering to the individual nHC-HA/PTX3 or rcHC-HA/PTX3complexes described herein by any suitable route of administration.Suitable methods for administration will depend on the disease orcondition to be treated. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complexes are administered locally to the site oftreatment. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complexes are administered systemically. Exemplary methods foradministration of the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes providedherein include but are not limited to parenteral, enteral, subcutaneous,percutaneous, transdermal, intradermal, intravenous, topical,inhalation, or implantation.

Scarring

Described herein, in certain embodiments, are methods of preventing,reducing, or reversing scarring in a subject in need thereof, comprisingadministering to the subject a composition comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein.

As used herein, “scarring” refers to the formation of a scar. In oneaspect, the scar is a hypertrophic scar, or keloid scar, or a scarresulting from acne. As used herein, a “scar” is an area of fibroustissue that results from the overproduction of collagen. In certaininstances, wound healing comprises the migration of fibroblasts to thesite of injury. In certain instances, fibroblasts deposit collagen. Incertain instances, fibroblasts deposit excess collagen at the woundsite, resulting in a scar.

In certain instances, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein prevents or inhibits TGF-β signaling. In certain instances, TGF-βregulates the extracellular matrix by stimulating fibroplasia andcollagen deposition and inhibiting extracellular matrix degradation (byup-regulating the synthesis of protease inhibitors). In certaininstances, preventing or inhibiting the expression of TGF- β results inthe prevention of or a reduction in intensity of a scar. In someembodiments, administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein prevents or reduces scarring.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein inhibits or prevents the ability of fibroblasts to differentiateinto myofibroblasts. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein reverts differentiated myofibroblasts tofibroblasts.

In some embodiments, a method disclosed herein is used to prevent,reduce or reverse the formation of a scar. In some embodiments, a methoddisclosed herein comprises administering an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein to an individual with a disorder that resultsin scarring (e.g., dermatitis scar, a keloid scar, contracture scar, ahypertrophic scar, or a scar resulting from acne). In some embodiments,a method disclosed herein comprises administering an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein to an individual in need thereofbefore or after trauma. In some embodiments, a method disclosed hereincomprises administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein to an individual in need thereof before or after surgery.

In some embodiments, a method disclosed herein is used to prevent orreduce the formation of a scar on an eye or on the surrounding tissue.In some embodiments, a method disclosed herein comprises administeringan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to an individualwith a disorder that results in scarring of the eye or surroundingtissue (e.g., retinopathy of prematurity). In some embodiments, a methoddisclosed herein comprises administering an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein to an individual in need thereof before orafter trauma to an eye or the surrounding tissue. In some embodiments, amethod disclosed herein comprises administering an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein to an individual in need thereofbefore or after surgery to an eye or the surrounding tissue.

Inflammation

Described herein, in certain embodiments, are methods of preventing orreducing inflammation in a subject in need thereof, comprisingadministering to the subject a composition comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein. As used herein, “inflammation”means physiological responses resulting from the migration of plasmaand/or leukocytes (e.g., lymphocytes, macrophages, granulocytes, andneutrophils) to the site of an infection or trauma (e.g., blunt forcetrauma, penetrating trauma, or surgery).

In certain instances, leukocytes secrete cytokines following contactwith an antigen. As used herein, “cytokines” are signaling proteins orglycoproteins. In certain instances, a cytokine binds to a cell-surfacereceptor. In certain instances, cytokines induces the chemotaxis ofleukocytes to the site of an infection. In certain instances, cellsurface receptors on a leukocyte detect chemical gradients of acytokine. In certain instances, a leukocyte follows the gradient to thesite of infection. In certain instances, the binding of a cytokine to acell-surface receptor results in the upregulation or downregulation ofcertain genes and their transcription factors. In certain instances,changes in gene expression results in the production of cytokines, anincrease in the production of cytokines, or an increase in thepresentation of cell surface receptors.

By way of non-limiting example, cytokines include interleukins IL-1,IL-6, IL-8, MCP-1 (also known as CCL2), and TNF-α. Interleukin 1 ispresent in the body in two isoforms: IL-1α and IL-1β. In certaininstances, the presence of IL-1 increases the expression of adhesionfactors on endothelial cells. This, in turn, enables the transmigrationof leukocytes to the site of infection. In certain instances, IL-8induces the chemotaxis of leukocytes. In certain instances, TNF-αinduces the chemotaxis of leukocytes. In certain instances, MCP-1recruits leukocytes to sites of tissue injury and infection.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein suppresses the production of and/or activity of cytokines. Incertain instances, a decrease in the concentration cytokines reduces orprevents inflammation by decreasing the number of leukocytes and/or therate at which leukocytes migrate to the site of an injury.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein induces apoptosis of a leukocyte (e.g., a macrophage, neutrophil,or lymphocyte). In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein decreases the number of activated leukocytes orthe rate at which leukocytes are activated. In certain instances, adecrease in the concentration of leukocytes reduces or preventsinflammation by decreasing the number (e.g., facilitate death of suchcells via apoptosis) of cells that migrate to the site of an injury.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein inhibits the polarization of a macrophage to an inflammatoryphenotype (i.e. an M1 phenotype). In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein reduces or inhibits the expressionof IL-12 or IL-23 in stimulated macrophages. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein promotes thepolarization of stimulated macrophages to a regulatory or wound healingM2 phenotype. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein inhibits or reduces inflammation in a subject.In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein inhibits or reduces tissue damage caused by an inflammatoryresponse in a subject. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex inhibits or reduces tissue damage caused by acondition or disease that induces inflammation in a subject.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having inflammation. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered to a subject having acute inflammation. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered to a subject having chronic inflammation. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered to a subject having an inflammatory disorder. In someembodiments, the inflammatory disorder is a macrophage mediatedinflammatory disorder. In some embodiments, the inflammatory disorder isa T-cell mediated inflammatory disorder. In some embodiments, theinflammatory disorder is a Th-17 mediated immune disorder.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having an acute inflammatoryresponse. In some embodiments, the acute inflammatory response is causedby, for example, an allergy, sepsis, endotoxic shock or ischemia, suchas but not limited to, myocardial infarction and stroke. In someembodiments, the acute inflammatory response is the result of bacterialinfection, a protozoal infection, a protozoal infection, a viralinfection, a fungal infection, or combinations thereof. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reducesacute inflammation. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3complex inhibits or reduces tissue damage caused by acute inflammation.In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits orreduces tissue reperfusion injury due to ischemia, including myocardialinfarction and stroke,

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having chronic inflammation that isassociated with the activation of lymphocytes via adaptive immunity. Insome embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having a Th1 response. In someembodiments the Th1 response leads to immune rejection of biologicaltransplant. In some embodiments, the transplant is an allografttransplant. In some embodiments, the transplant is an autologoustransplant. In some embodiments, the inflammatory disorder is graftversus host disease or tissue transplant rejection. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex inhibits or reduces chronicinflammation in a subject.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having chronic inflammation that isassociated with a Th17 immune response associated with an inflammatorydisorder. In some embodiments, the inflammatory disorder is anautoimmune disorder or a leukocyte defect.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a subject having an inflammatory disorder thatis acute disseminated encephalomyelitis; Addison’s disease; ankylosingspondylitis; antiphospholipid antibody syndrome; autoimmune hemolyticanemia; autoimmune hepatitis; autoimmune inner ear disease; bullouspemphigoid; Chagas disease; chronic obstructive pulmonary disease;coeliac disease; dermatomyositis; diabetes mellitus type 1; diabetesmellitus type 2; endometriosis; Goodpasture’s syndrome; Graves’ disease;Guillain-Barré syndrome; Hashimoto’s disease; idiopathicthrombocytopenic purpura; interstitial cystitis; systemic lupuserythematosus (SLE); metabolic syndrome, multiple sclerosis; myastheniagravis; myocarditis, narcolepsy; obesity; pemphigus vulgaris; perniciousanemia; polymyositis; primary biliary cirrhosis; rheumatoid arthritis;schizophrenia; scleroderma; Sjögren’s syndrome; vasculitis; vitiligo;Wegener’s granulomatosis; allergic rhinitis; prostate cancer; non-smallcell lung carcinoma; ovarian cancer; breast cancer; melanoma; gastriccancer; colorectal cancer; brain cancer; metastatic bone disorder;pancreatic cancer; a lymphoma; nasal polyps; gastrointestinal cancer;ulcerative colitis; Crohn’s disorder; collagenous colitis; lymphocyticcolitis; ischaemic colitis; diversion colitis; Behçet’s syndrome;infective colitis; indeterminate colitis; inflammatory liver disorder,ischemia, myocardial infarction, stroke, endotoxin shock, septic shock;rheumatoid spondylitis, ankylosing spondylitis, Gouty arthritis,polymyalgia rheumatica, Alzheimer’s disorder, Parkinson’s disorder,epilepsy, AIDS dementia, asthma, adult respiratory distress syndrome,bronchitis, cystic fibrosis, acute leukocyte-mediated lung injury,distal proctitis, Wegener’s granulomatosis, fibromyalgia, uveitis,conjunctivitis, psoriasis, eczema, dermatitis, smooth muscleproliferation disorders, meningitis, shingles, encephalitis, nephritis,tuberculosis, retinitis, atopic dermatitis, pancreatitis, periodontalgingivitis, coagulative Necrosis, liquefactive necrosis, fibrinoidnecrosis, neointimal hyperplasia, or combinations thereof.

In some embodiments, the inflammatory disorder is an inflammatorydisorder of an eye or the surrounding tissue. In some embodiments, theinflammatory disorder is conjunctivitis. In certain instances,conjunctivitis results from exposure to an allergen. In certaininstances, conjunctivitis results from a bacterial infection. In someembodiments, the inflammatory disorder is keratitis. As used herein,“keratitis” is a disorder characterized by inflammation of the cornea.In some embodiments, the inflammatory disorder is keratoconjunctivitis(i.e., a combination of conjunctivitis and keratitis (i.e., cornealinflammation)). In some embodiments, the inflammatory disorder isblepharitis. As used herein, “blepharitis” is an ophthalmic disordercharacterized by inflammation of the eyelid margins. In someembodiments, the inflammatory disorder is blepharoconjunctivitis (i.e.,a combination of conjunctivitis and blepharitis (i.e., inflammation ofan eyelid)). In some embodiments, the inflammatory disorder isscleritis. As used herein, “scleritis” is a disorder characterized byinflammation of the sclera. In some embodiments, the inflammatorydisorder is episcleritis. As used herein, “episcleritis” is aninflammatory disorder of the episclera characterized by hyperaemia, andchemosis. In some embodiments, the inflammatory disorder is uveitis. Asused herein, “uveitis” is an inflammatory disorder of the uvea. In someembodiments, the disorder is retinitis. As used herein, “retinitis” isan inflammatory disorder of a retina. In some embodiments, the disorderis choroiditis. As used herein, “choroiditis” is an inflammatorydisorder of the uvea, ciliary body and the choroid.

Abnormal Angiogenesis

Disclosed herein, in certain embodiments, are methods of preventing orreducing angiogenesis in a subject in need thereof, comprisingadministering to the subject a composition comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein. As used herein, “angiogenesis”means the formation of new blood vessels. In certain instances,angiogenesis facilitates the growth and metastasis of a tumor. Further,in certain instances, abnormal angiogenesis is the basis of wetage-related macular degeneration (wARMD) and diabetic proliferativeretinopathy. In certain instances, an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein prevents or reduces angiogenesis.

In certain instances, the binding of a ligand to the VEGF receptor-2(VEGFR-2) starts a tyrosine kinase signaling cascade that stimulates theproduction of factors that variously stimulate vessel permeability(eNOS, producing NO), proliferation/survival (bFGF), migration(ICAMs/VCAMs/MMPs) and finally differentiation into mature bloodvessels. In certain instances, following binding of VEGFR-2 to itsligand, endothelial cells form tube structures resembling capillaries.

As used herein, “wet Age Related Macular Degeneration”, “wARMD”, or “wetARMD” means a disorder of an eye characterized by the proliferation ofblood vessels from the choroid. In certain instances, wet ARMD causesvision loss due blood and protein leakage below the macula. In certaininstances, bleeding, leaking, and scarring from these blood vesselscause irreversible damage to the photoreceptors and rapid vision loss ifleft untreated.

As used herein, “diabetic proliferative retinopathy” means a disorder ofan eye characterized by incompetence of the vascular walls. In certaininstances, the lack of oxygen in the retina results in angiogenesisalong the retina and in the vitreous humour. In certain instances, thenew blood vessels bleed, cloud vision, and destroy the retina.

In certain instances, the proliferation of capillaries supplies a tumorwith nutrients, allowing the tumor to expand. In certain instances, theproliferation of capillaries enables the rapid removal of cellular wasteenabling tumor growth. In certain instances, angiogenesis facilitatesmetastasis. In certain instances, the proliferation of capillariesincreases the chances that a cancerous cell will be able to enter ablood vessel and thus establish a new tumor at a new site.

Exemplary cancer types that are treated in some embodiments using annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein include, but arenot limited to, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,Adrenocortical Carcinoma, AIDS-Related Cancers, AIDS-Related Lymphoma,Anal Cancer, Astrocytoma, Basal Cell Carcinoma, Bile Duct Cancer,Bladder Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, BrainTumor, Breast Cancer, Bronchial Adenomas, Burkitt’s Lymphoma, CarcinoidTumor, Carcinoma, Central Nervous System Lymphoma, CerebellarAstrocytoma, Cervical Cancer, Chronic Lymphocytic Leukemia, ChronicMyelogenous Leukemia, Chronic Myeloproliferative Disorders, ColonCancer, Colorectal Cancer, Cutaneous T-Cell Lymphoma, EndometrialCancer, Ependymoma, Esophageal Cancer, Extragonadal Germ Cell Tumor, EyeCancer, Intraocular Melanoma, Eye Cancer, Retinoblastoma, GallbladderCancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor(GIST), Germ Cell Tumor (Extracranial), Germ Cell Tumor (Extragonadal),Germ Cell Tumor (Ovarian), Gestational Trophoblastic Tumor, Glioma,Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular (Liver)Cancer, Hodgkin’s Lymphoma, Hypopharyngeal Cancer, Hypothalamic andVisual Pathway Glioma, Intraocular Melanoma, Islet Cell Carcinoma(Endocrine Pancreas), Kaposi’s Sarcoma, Kidney (Renal Cell) Cancer,Laryngeal Cancer, Leukemia (Acute Lymphoblastic), Leukemia (AcuteMyeloid), Leukemia (Chronic Lymphocytic), Leukemia (ChronicMyelogenous), Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer(Non-Small Cell), Lung Cancer (Small Cell), Lymphoma, (CutaneousT-Cell), Lymphoma (Non-Hodgkin’s), Malignant Fibrous Histiocytoma ofBone/Osteosarcoma, Medulloblastoma, Melanoma, Merkel Cell Carcinoma,Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary,Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma CellNeoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia,Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity andParanasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, OralCancer, Oropharyngeal Cancer, Osteosarcoma/Malignant FibrousHistiocytoma of Bone, Ovarian Cancer, Ovarian Epithelial Cancer, OvarianGerm Cell Tumor, Ovarian Low Malignant Potential Tumor, PancreaticCancer, Parathyroid Cancer, Penile Cancer, Pheochromocytoma,Pineoblastoma and Supratentorial Primitive Neuroectodermal Tumors,Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, PleuropulmonaryBlastoma, Prostate Cancer, Rectal Cancer, Retinoblastoma,Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoma (Kaposi’s), Sarcoma(uterine), Sezary Syndrome, Skin Cancer (non-Melanoma), Skin Cancer(Melanoma), Skin Carcinoma (Merkel Cell), Small Intestine Cancer, SoftTissue Sarcoma, Squamous Cell Carcinoma, Stomach (Gastric) Cancer,T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer,Trophoblastic Tumor, Gestational, Urethral Cancer, Uterine Cancer,Endometrial, Uterine Sarcoma, Vaginal Cancer, Visual Pathway andHypothalamic Glioma, Vulvar Cancer, Waldenström’s Macroglobulinemia, andWilms’ Tumor.

Wound Repair and Tissue Regeneration

In some embodiments, a pharmaceutical compositions containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a woundcovering or is used to facilitate wound repair. In some embodiments, thetissue was damaged, compromised, or lost due to an injury (e.g., a burn;a surgical incision; an area of necrosis resulting from an infection,trauma, or a toxin; a laceration). In some embodiments, the tissue wasdamaged, compromised, or lost due to a burn. In some embodiments, thetissue was damaged, compromised, or lost due to a wound (e.g., anincision, laceration, abrasion). In some embodiments, the tissue wasdamaged, compromised, or lost due to necrosis. In some embodiments, thetissue was damaged, compromised, or lost due to ulceration.

Burns

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to aburn. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to afirst degree burn. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a second degree burn. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to a third degree burn. In some embodiments, thepharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex is applied to a substrate prior to be placed on the burn.

Wounds

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to awound in the skin (e.g., an incision, laceration, abrasion, ulcer,puncture, penetration). In some embodiments, the wound is an ischemicwound. In some embodiments, the pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrate prior tobeing placed on the wound. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein treats the wound.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to anincision in an organ (e.g., the skin, brain, stomach, kidneys, liver,intestines, lungs, bladder, trachea, esophagus, vagina, ureter, andblood vessel walls). In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a surgical incision. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to the site of a colon resection. In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to the site of a gastrectomy. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is applied to the site of abreast surgery (e.g., breast reduction surgery, breast augmentationsurgery, and mastectomy). In some embodiments, the pharmaceuticalcomposition containing nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied toa substrate prior to being placed on the wound.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as acovering over an incision in the skin (e.g., an incision to theepidermis, dermis, and/or hypodermis). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is used to repair or supplement the skinfollowing hemorrhoid surgery. In some embodiments, the pharmaceuticalcomposition containing nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied toa substrate prior to being placed on the wound. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein treats the wound.

Necrosis

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as aprotective graft over an area of necrotic tissue (e.g., from aninfection). In some embodiments, a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as aprotective graft over an area of necrotic skin. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is placed on an area of necrotic tissue. Insome embodiments, the pharmaceutical composition containing nHC-HA/PTX3or rcHC-HA/PTX3 complex is applied to a substrate prior to being placedon the necrotic tissue. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein treats the necrotic tissue.

Ulcer

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as aprotective covering over an ulcer. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein treats the ulcer. In some embodiments, the ulcer is a diabeticulcer, such as a diabetic foot or leg ulcer. In some embodiments, theulcer is an ischemic wound. In some embodiments, the pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is appliedto a substrate prior to being placed on the ulcer. In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein treats the ulcer. In some embodiments the ulceris a non-healing ulcer. For example, in some embodiments thatnon-healing ulcer is a wound or ulcer on the skin that has been presentfor about 3-4 weeks duration without healing.

In some embodiments, the ulcer is a foot ulcer (e.g., a diabetic footulcer or an arterial insufficiency ulcer). In some embodiments, treatinga foot ulcer comprises (a) preparing the wound (e.g., debriding thewound); and (b) placing a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound. Insome embodiments, treating a foot ulcer comprises (a) preparing thewound (e.g., debriding the wound); (b) placing a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein on the wound; and (c) covering the pharmaceutical compositionwith a protective barrier (e.g., a silvercell dressing, metipel, gauze,or a bandage). In some embodiments, the pharmaceutical compositioncontaining nHC-HA/PTX3 or rcHC-HA/PTX3 complex is applied to a substrateprior to be placed on the ulcer. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein treats the ulcer.

In some embodiments, the ulcer is a venous stasis (VS) ulcer. In someembodiments, treating a VS ulcer comprises (a) preparing the wound(e.g., debriding the wound); and (b) placing a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein on the wound. In some embodiments, treating a VS ulcer comprises(a) preparing the wound (e.g., debriding the wound); (b) placing apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein on the wound; and (c) covering thepharmaceutical composition with a protective barrier (e.g., a woundveil, antimicrobial dressing, gauze, or a bandage). In some embodiments,the pharmaceutical composition containing nHC-HA/PTX3 or rcHC-HA/PTX3complex is applied to a substrate prior to being placed on the wound. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein treats the ulcer.

In some embodiments, the ulcer is a corneal ulcer (i.e., ulcerativekeratitis). In some embodiments, treating a corneal ulcer comprises (a)preparing the wound (e.g., debriding the wound); and (b) placing apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein on the wound. In some embodiments, treating acorneal ulcer comprises (a) preparing the wound (e.g., debriding thewound); (b) placing a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein on the wound; and(c) covering the pharmaceutical composition with a protective barrier(e.g., a contact lens or a bandage). In some embodiments, thepharmaceutical composition containing nHC-HA/PTX3 or rcHC-HA/PTX3complex is applied to a substrate prior to being placed on the wound. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein treats the ulcer.

Therapeutic Cell Therapies

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered in combination with a cell therapy. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isco-administered with a therapeutic cell. Therapeutic cells include anycell that exhibits a therapeutic property for treatment of a disease ordisorder. In some embodiments, the therapeutic cell is a recombinantcell that heterologously expresses one or more therapeutic geneproducts. In some embodiments, the therapeutic cell is a transplantedcell. In some embodiments, the therapeutic cell is a stem cell. In someembodiments, the therapeutic cell is a cell that expresses one or morestem cell markers (e.g. Oct-¾ (Pou5f1), Sox2, c-Myc, and Klf4).

In some embodiments, the cell therapy is a stem cell transplant. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is administered topromote expansion of stem cells of the transplant and tissueregeneration. In some examples, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexis employed to reduces or inhibit inflammation, scarring, and abnormalangiogenesis caused by a stem cell transplant. In some embodiments,nHC-HA/PTX3 or rcHC-HA/PTX3 complex is employed to maintain the stemcell characteristics during ex vivo expansion by substituting feederlayers. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 complex isemployed to reprogram a differentiated cell to a stem cell. In someembodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isemployed to expand and culture stem cells in vitro for subsequenttransplant into a subject.

In some embodiments, the stem cell therapy is an embryonic stem celltherapy. In some embodiments, the stem cell therapy is an adult stemcell therapy. In some embodiments, the stem cell therapy is amesenchymal stem cell therapy. In some embodiments, the stem celltherapy is administered for the treatment of a disease or disorder, suchas, but not limited to, cardiovascular disease, cancer, diabetes, spinalcord injury, neurodegenerative disease, Alzheimer’s disease, Parkinson’sdisease, multiple sclerosis, Amytrophic lateral sclerosis, DuchenneMuscular Dystrophy, muscle damage or dystrophy, stroke, burns, lungdisease, retinal disease, kidney disease, osteoarthritis, and rheumatoidarthritis.

In some embodiments, stem cells are used to treat diabetes mellitus.Type 1 diabetes results from autoimmune-mediated destruction ofinsulin-secreting β cells in the islets of Langerhans of the pancreas.Type 2 diabetes results from systemic insulin resistance and reducedinsulin secretion by pancreatic β cells. Stem cells have been shown invitro to differentiate into insulin-producing cells (see e.g.,Schuldiner et al. (2000) Proc. Natl. Acad. Sci. USA. 97: 11307-11312;Guo et al., (2009) Endocr Rev 30:214-227). Thus, in some embodiments,stem cells, including ESCs and ASCs, and their derivatives, such aspartially differentiated stem cells, are used in stem cell therapy forregeneration of pancreatic β cells.

In some embodiments, stem cells or differentiated cells employed fortherapy are encapsulated in a microcapsule device. In some embodiments,the microcapsule comprises an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein. In some embodiments the nHC-HA/PTX3 or rcHC-HA/PTX3complex is covalently attached to the microcapsule. In some embodimentsthe nHC-HA/PTX3 or rcHC-HA/PTX3 complex is assembled on the surface ofthe microcapsule, such as the inner or out surface, or both. In someembodiments the nHC-HA/PTX3 or rcHC-HA/PTX3 is formulated to coat themicrocapsule. In some embodiments, the microcapsule comprises pores toallow the passage of nutrients to cells into the microcapsule and/orallows secreted proteins and molecules (e.g., insulin) by theencapsulated cells to flow out of the microcapsule. In some embodiments,the cells are first immobilized on a microcarrier, such as a bead coatedwith Matrigel® and then encapsulated within the microcapsule. Methodsfor the encapsulation of cells, such as stem cells are known in the artand are described, for example, in Serra et al. (2011) PLoS ONE6(8):e23212. In some embodiments, any method for the encapsulation ofcells is employed in conjunction with the methods provided herein.

In some embodiments, allogeneic therapeutic stem cells (e.g., insulinproducing islet cells) are encapsulated in a microcapsule device forproduction of insulin. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complex promotes the expansion of the stem cells. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex, inhibits orreduces an inflammatory response against the microcapsule containing thestem cells employed for therapy of diabetes mellitus. In someembodiments, the micro capsule comprises an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complex inhibits or reduces an inflammatory responseagainst the microcapsule containing the stem cells.

Soft Tissue Uses

Disclosed herein, in certain embodiments, is the use of an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein for repairing, reconstructing,replacing, or supplementing a recipient’s damaged, compromised, ormissing soft tissue (e.g., tendons). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied directly to the tissue. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is administered in conjunctionwith cell or tissue based therapies. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is mixed with a cell, a plurality of cells, ora tissue and is administered as part of a tissue based therapy. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is used to coat a cell, aplurality of cells, or a tissue and is administered as part of a tissuebased therapy.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as acovering over an incision in soft tissue (e.g., eyelids form the tissueplane between different layers of soft tissue). In some embodiments, thepharmaceutical composition containing nHC-HA/PTX3 or rcHC-HA/PTX3complex is applied to a substrate and then used as a covering over anincision in soft tissue (e.g., eyelids form the tissue plane betweendifferent layers of soft tissue).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used asstructural (tectonic) support for soft tissue. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein prevents adhesion in joint or tendon repairs.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used in therepair a tendon or joint (such as rotator cuff repairs, hand tendonrepairs). In some embodiments, a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used toreinforce a tendon or joint. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is used to prevent adhesion of a healing tendon to surroundingtissue, tendons or joints. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is used to prevent the formation of scar tissue on a tendon.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to augment smaller tendons and ligaments of the foot andankle, including the posterior tibial tendon, the peroneal tendons, theflexor and extensor tendons, and the ligaments of the lateral anklecomplex. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to reinforce primary repair of the quadriceps andpatellar tendons surrounding the knee. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as aperiosteal patch for bone graft in joint replacement. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used toaugment deficient hip and knee capsular tissue following total jointrevision surgery.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used in the repair of a torn rotator cuff. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as apatch over a rotator cuff muscle or tendon (e.g., the supraspinatustendon). In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to reconstruct a rotator cuff muscle or tendon (e.g.,the supraspinatus tendon). In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used to augment a rotator cuff muscleor tendon (e.g., the supraspinatus tendon). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used toreinforce a rotator cuff muscle or tendon (e.g., the supraspinatustendon). In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to prevent adhesion of soft tissue to a rotator cuffmuscle or tendon (e.g., the supraspinatus tendon).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used in therepair gingiva. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused in the repair gingival recession. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and used as a patchover gingiva. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to substrate and used as a patch over an exposed tooth rootsurface. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used toreconstruct gingiva. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused to augment gingiva. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is used to reinforce gingiva. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is used to prevent adhesion of soft tissue togingiva.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a protective graft over an incision or tear in thefascia. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as structural (tectonic) support the fascia. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement or supplement for the fascia. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used torepair a hernia (e.g., to repair the fascia). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/ rcHC-HA/PTX3 complex is used torepair an inguinal hernia. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used to repair a femoral hernia. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate andthe substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used torepair an umbilical hernia. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used to repair an incisional hernia.In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to repair a diaphragmatic hernia. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used torepair a Cooper’s hernia, an epigastric hernia,an hiatal hernia, aLittre’s hernia, a lumbar hernia, a Maydl’s hernia, an obturator hernia,a pantaloon hernia, a paraesophageal hernia, a paraumbilical hernia, aperineal hernia, a properitoneal hernia, a Richter’s hernia, a slidinghernia, a sciatic hernia, a spigelian hernia, a sports hernia, a Velpeauhernia, or a Amyand’s hernia.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to repair a spinal disc herniation. In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as aprotective graft over an incision or tear in a spinal disc. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as aprotective graft over an incision or tear in an annulus fibrosis. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate andthe substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support a spinal disc. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support an annulus fibrosis. In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement or supplement for a spinal disc. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support a spinal disc. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement or supplement for an annulus fibrosis.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used over an incision in the brain, or in one (or all) of themeninges (i.e., the dura mater, the pia mater, and/or the arachnoidmater). In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as structural (tectonic) support for one (or all) of themeninges (i.e., the dura mater, the pia mater, and/or the arachnoidmater). In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a replacement for one (or all) of the meninges (i.e.,the dura mater, the pia mater, and/or the arachnoid mater).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used over an incision in a lung or in the pleura. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for the pleura. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement for the pleura.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used over an incision in a tympanic membrane. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for a tympanic membrane. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement for a tympanic membrane.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a protective graft over an incision in the heart orthe pericardium. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used as structural (tectonic) supportfor the pericardium. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used as a replacement for thepericardium.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a protective graft over an incision in theperitoneum. In some embodiments, a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as structural (tectonic) support for the peritoneum. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate andthe substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement for the peritoneum.

Ophthalmic Uses

Disclosed herein, in certain embodiments, is the use of a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein for repairing, reconstructing, replacing, or supplementing arecipient’s damaged, compromised, or missing ocular tissue.

Treatment of Glaucoma

As used herein, “Glaucoma” means a disorder characterized by the loss ofretinal ganglion cells in the optic nerve. In certain instances,glaucoma partially or fully results from an increase in intraocularpressure in the anterior chamber (AC). Intraocular pressure variesdepending on the production of liquid aqueous humor by the ciliaryprocesses of the eye and the drainage of the aqueous humor through thetrabecular meshwork.

Glaucoma Drainage Devices (GDD) are medical devices that are implantedinto an eye to relieve intraocular pressure by providing an alternativepathway for the aqueous humor to drain. If left uncovered, a GDD tubewill erode and leave the eye susceptible to intraocular infection. Thus,the GDD tube needs to be covered. Currently, patches used to cover GDDtubes are made from pericardium, sclera and cornea. These patches areabout 400-550 microns thick. The thinness of these patches results intheir melting by 25% in 2 years potentially leaving the shunt tubeexposed again.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to cover GDD tubes. In some embodiments, thesubstrate/nHC-HA/PTX3 or rcHC-HA/PTX3 complex is 300-600 microns thick.In some embodiments, the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex does not melt by 25% in 2 years.

Treatment of Ocular Ulcers

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used to cover persistent epithelial defects and/or ulcers ineyes.

In some embodiments, the base of the ulcer is debrided with surgicalsponges and the poorly adherent epithelium adjacent to the edge of theulcer is removed (e.g., to the section of the eye where the epitheliumbecomes quite adherent). In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is applied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is transferred to the recipient eye. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein is applied to a substrate andthe substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is thensecured to the eye by sutures (e.g., interrupted 10-0 nylon sutures orrunning 10-0 nylon sutures) with the suture knots being buried. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is secured tothe eye by use of fibrin glue. In some embodiments, a protective layeris applied over the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex or the entire eye (e.g., a contact lens). In some embodiments,the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex furthercomprises an antibiotic (e.g., neomycin, polymyxin b sulfate anddexamethasone).

Conjunctival, Scleral, Lid, and Orbital Rim Surface Reconstruction

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used in conjunctival, scleral, lid, and orbital rim surfacereconstruction. In some embodiments, damage to the conjunctival surfaceresults from symblepharon lysis; surgical removal of tumor, lesion,and/or scar tissue; excimer laser photorefractive keratectomy andtherapeutic keratectomy; or combinations thereof.

Coronary Uses

Disclosed herein, in certain embodiments, is the use of a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein for repairing, reconstructing, replacing, or supplementing arecipient’s damaged, compromised, or missing coronary tissue.

Prevention of Ischemia Reperfusion Damage

Disclosed herein, is the use of a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein for theinhibition or reduction of tissue damage resulting from acuteinflammation caused by ischemia, such as, for example myocardialinfarction or stroke. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isadministered to a subject having an ischemic condition, such as, but notlimited to myocardial infarction or stroke.

Coronary Artery Bypass

Disclosed herein, is the use of a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein in coronaryartery bypass surgery. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is grafted onto a coronary artery tobypass a section of the artery that is characterized by atherosclerosis.

Heart Valves

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is applied over a heart valve. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for a heart valve. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement for a heart valve.

Veins and Arteries

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is applied to a vein or artery. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for a vein or artery.

Nerve Uses

Disclosed herein, in certain embodiments, is the use of a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein for repairing, reconstructing, replacing, or supplementing arecipient’s damaged, compromised, or missing nerve tissue.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a covering over a nerve (e.g., a peripheral nerve).In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a covering over a nerve graft, nerve transfer, or arepaired nerve. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isapplied to a substrate and the substrate/nHC-HA/PTX3 orsubstrate/rcHC-HA/PTX3 complex is used as a covering over an incision ina nerve (e.g., a peripheral nerve). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for a nerve (e.g., a peripheral nerve). Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex disclosed herein prevents adhesion in nerverepair.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to asubstrate and the substrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3complex is used as a non-constricting encasement for injured nerves. Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex described herein prevents or minimizes scarformation, encapsulation, chronic compression, tethering of a nerve, andnerve entrapment. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described hereinprevents or minimizes neuroma formation. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein prevents or minimizes the migration ofendogenous growth factors (i.e. Nerve Growth Factor) present duringnerve repair.

Spinal Uses

Disclosed herein, in certain embodiments, is the use of a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex describedherein during spinal surgery.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used during alaminectomy. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein isused to reduce or prevent epidural fibrosis and/or scar adhesionsfollowing spinal surgery (e.g., laminectomy). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein is implanted between dura mater and overlyingtissue following spinal surgery (e.g., laminectomy). In someembodiments, implanting a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein between dura materand overlying tissue following spinal surgery (e.g., laminectomy)reduces or prevents migration of fibroblasts to the dura mater andcollagen deposition on the dura mater.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduceor prevent the development of proliferative scarring following spinalsurgery (e.g., laminectomy). In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex describedherein is used to reduce or prevent the development of a postoperative(e.g., postlaminectomy) epidural/peridural/perineural scar. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex described herein is used to reduce or prevent thedevelopment of proliferative scarring following spinal surgery (e.g.,laminectomy). In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused to reduce or prevent the development of a postlaminectomy membrane.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex described herein is used to reduceor prevent the development of extradural compression or dural tetheringfollowing spinal surgery (e.g., laminectomy). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex described herein is used to reduce or prevent the development oftethered nerve roots following spinal surgery (e.g., laminectomy). Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex described herein is used to reduce or preventthe development of arachnoiditis following spinal surgery (e.g.,laminectomy).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein further comprisesmorselized bone tissue. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein comprising morselized bone tissue is used during a spinal fusionprocedure. In some embodiments, a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein comprisingmorselized bone tissue is implanted between adjacent vertebrae. In someembodiments, implantation of a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein comprisingmorselized bone tissue between two adjacent vertebrae promotes fusion ofthe vertebrae.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as aprotective graft over an incision in the dura mater. In someembodiments, a pharmaceutical composition containing an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used asstructural (tectonic) support for the dura mater. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is applied to a substrate and thesubstrate/nHC-HA/PTX3 or substrate/rcHC-HA/PTX3 complex is used as areplacement for the dura mater.

Miscellaneous Uses of an nHC-HA/PTX3 or rcHC-HA/PTX3 Complex

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is applied to apatch or wound dressing.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used as a dermalfiller. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is injected intosubdermal facial tissues. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is injected under wrinkles and aging lines of the face (e.g.,nasolabial folds, melomental folds, “crow’s feet” and foreheadwrinkles). In some embodiments, a pharmaceutical composition containingan nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used for lipaugmentation. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isinjected into the lips.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treatarthritis (e.g., osteoarthritis, rheumatoid arthritis, septic arthritis,ankylosing spondylitis, spondylosis). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein is injected into an arthritic joint (e.g., aknee).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treatarthritis in the foot. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused to treat arthritis of the first metatarsophalangeal (MTP) joint(e.g., hallux rigidus). In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered to a MTP joint following dorsal cheilectomy. Insome embodiments, administration of the pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed hereinreduces one or more adverse symptoms associated with hallux rigidus or adorsal cheilectomy procedure (e.g., scarring, joint stiffness, swelling,inflammation, and pain).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treatone or more symptoms associated with a bone spur (e.g., scarring, jointstiffness, swelling, inflammation, and pain).

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to inhibitbone resorption in an individual in need thereof. In some embodiments,the individual has arthritis, osteoporosis, alveolar bone degradation,Paget’s disease, or a bone tumor. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex isinjected into a joint. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with abone (e.g., by use of a wound dressing or bandage). In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex coats a bone stent, bone implant, or bone prosthesis (e.g., anosseointegrated implant). As used herein, an “osseointegrated implant”means a three dimensional implant containing pores into whichosteoblasts and supporting connective tissue migrates. In someembodiments, the bone stents are inserted into the intramedullary canalof a bone. In some embodiments, the bone stent is placed in the sinustarsi. In some embodiments, the bone stent in placed in a knee or joint.In some embodiments, the bone stent is placed in a bone fracture. Insome embodiments, the bone stent is expandable or contractible.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to promoteor induce bone formation in an individual in need thereof in anindividual in need thereof. In some embodiments, the individual hasarthritis, osteoporosis, alveolar bone degradation, Paget’s disease, ora bone tumor. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into ajoint. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with a bone (e.g., byuse of a wound dressing or bandage). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex coats a bone stent, bone implant, or bone prosthesis (e.g., anosseointegrated implant). As used herein, an “osseointegrated implant”means a three dimensional implant containing pores into whichosteoblasts and supporting connective tissue migrates. In someembodiments, the bone stents are inserted into the intramedullary canalof a bone. In some embodiments, the bone stent is placed in the sinustarsi. In some embodiments, the bone stent in placed in a knee or joint.In some embodiments, the bone stent is placed in a bone fracture. Insome embodiments, the bone stent is expandable or contractible.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to inhibitosteoclast differentiation. In some embodiments, the individual hasarthritis, osteoporosis, alveolar bone degradation, Paget’s disease, ora bone tumor. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex is injected into ajoint. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex is contacted with a bone (e.g., byuse of a wound dressing or bandage). In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex coats a bone stent, bone implant, or bone prosthesis (e.g., anosseointegrated implant). As used herein, an “osseointegrated implant”means a three dimensional implant containing pores into whichosteoblasts and supporting connective tissue migrates. In someembodiments, the bone stents are inserted into the intramedullary canalof a bone. In some embodiments, the bone stent is placed in the sinustarsi. In some embodiments, the bone stent in placed in a knee or joint.In some embodiments, the bone stent is placed in a bone fracture. Insome embodiments, the bone stent is expandable or contractible.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to promotemineralization by osteoblasts in an individual in need thereof. In someembodiments, the individual has arthritis, osteoporosis, alveolar bonedegradation, Paget’s disease, or a bone tumor. In some embodiments, apharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex is injected into a joint. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex iscontacted with a bone (e.g., by use of a wound dressing or bandage). Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex coats a bone stent, bone implant, or boneprosthesis (e.g., an osseointegrated implant). As used herein, an“osseointegrated implant” means a three dimensional implant containingpores into which osteoblasts and supporting connective tissue migrates.In some embodiments, the bone stents are inserted into theintramedullary canal of a bone. In some embodiments, the bone stent isplaced in the sinus tarsi. In some embodiments, the bone stent in placedin a knee or joint. In some embodiments, the bone stent is placed in abone fracture. In some embodiments, the bone stent is expandable orcontractible.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to balancebone resorption and bone formation in an individual in need thereof. Insome embodiments, the individual has arthritis, osteoporosis, alveolarbone degradation, Paget’s disease, or a bone tumor. In some embodiments,a pharmaceutical composition containing an nHC-HA/PTX3 or rcHC-HA/PTX3complex is injected into a joint. In some embodiments, a pharmaceuticalcomposition containing an nHC-HA/PTX3 or rcHC-HA/PTX3 complex iscontacted with a bone (e.g., by use of a wound dressing or bandage). Insome embodiments, a pharmaceutical composition containing an nHC-HA/PTX3or rcHC-HA/PTX3 complex coats a bone stent, bone implant, or boneprosthesis (e.g., an osseointegrated implant). As used herein, an“osseointegrated implant” means a three dimensional implant containingpores into which osteoblasts and supporting connective tissue migrates.In some embodiments, the bone stents are inserted into theintramedullary canal of a bone. In some embodiments, the bone stent isplaced in the sinus tarsi. In some embodiments, the bone stent in placedin a knee or joint. In some embodiments, the bone stent is placed in abone fracture. In some embodiments, the bone stent is expandable orcontractible.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used to treat anorthodontic or a periodontal condition. In some embodiments, theperiodontal condition is selected from gingivitis, gingival recession orperiodontitis. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused as an anti-inflammatory or used to promote osseointegration orhealing. In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is used incombination with a dental implant to promote implant osseointegration,anti-inflammation, and healing.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein to treat hoarsenessor voice disorders. In some embodiments, a pharmaceutical compositioncontaining an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isused for injection laryngoplasty to repair vocal cords.

In some embodiments, a pharmaceutical composition containing annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is coated onto amedical implant (e.g., a stent). In some embodiments, a medicalimplant/nHC-HA/PTX3 or implant/rcHC-HA/PTX3 complex disclosed herein isimplanted into an individual in need thereof, wherein the nHC-HA/PTX3 orrcHC-HA/PTX3 complex is partially or fully released into the individual.In some embodiments, the medical implant is a stent (e.g., a bone stentor a coronary stent). In some embodiments, the medical implant is a bonestent. In some embodiments, the medical implant is a coronary stent.

Combinations

In some embodiments, the compositions and methods described herein areused in conjunction with a second therapeutic agent. In someembodiments, the compositions and methods described herein are used inconjunction with two or more therapeutic agents. In some embodiments,the compositions and methods described herein are used in conjunctionwith one or more therapeutic agents. In some embodiments, thecompositions and methods described herein are used in conjunction with2, 3, 4, 5, 6, 7, 8, 9, 10 or more therapeutic agents.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein and a second therapeutic agent are administered in the samedosage form. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein and a second therapeutic agent are administered inseparate dosage forms.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein and a second therapeutic agent are administered concurrently(e.g., simultaneously, essentially simultaneously or within the sametreatment protocol).

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein and a second therapeutic agent are administered sequentially. Insome embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered before or after the second therapeutic agent. Insome embodiments, the time period between administration of annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second activeagent ranges from a few minutes to several hours, depending upon theproperties of each pharmaceutical agent, such as potency, solubility,bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent. In some embodiments, circadian variation of thetarget molecule concentration determines the optimal dose interval. Insome embodiments, the timing between the administration of annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second activeagent is about an hour, about 2 hours, about 3 hours, about 4 hours,about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9hours, about 10 hours, about 11 hours, about a day, about 2 days, about3 days, about 4 days, about 5 days, about 6 days, about a week, about 2weeks, about 3 weeks, about a month, or longer.

In some embodiments, the co-administration of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein results in a lower required dosagefor the nHC-HA/PTX3 or rcHC-HA/PTX3 complex than the required dosagewhen administering an nHC-HA/PTX3 or rcHC-HA/PTX3 complex alone. In someembodiments, the co-administration of a second therapeutic agent resultsin a lower required dosage for the second agent than the required dosagewhen administering the second agent alone. Methods for experimentallydetermining therapeutically-effective dosages of drugs and other agentsfor use in combination treatment regimens are known and described in theart. For example, the use of metronomic dosing, i.e., providing morefrequent, lower doses in order to minimize toxic side effects, has beendescribed extensively in the art. Combination treatment further includesperiodic treatments that start and stop at various times to assist withthe clinical management of the individual.

In some embodiments, the combination treatment nHC-HA/PTX3 orrcHC-HA/PTX3 complex and one or more additional therapeutic agents ismodified. In some embodiments, the combination treatment is modified,whereby the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex isincreased relative to the amount of a second therapeutic agent. In someembodiments, the combination treatment is modified, whereby the amountof the nHC-HA/PTX3 or rcHC-HA/PTX3 complex is decreased relative to theamount of a second therapeutic agent. In some embodiments, thecombination treatment is modified, whereby the amount of is a secondtherapeutic agent increased relative to the amount of the nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, the combination treatment ismodified, whereby the amount of is a second therapeutic agent decreasedrelative to the amount of the nHC-HA/PTX3 or rcHC-HA/PTX3 complex.

In some embodiments, the second therapeutic agent is selected fromcytotoxic agents, an antimicrobial agents, anti-angiogenesis agents, achemotherapeutic agent, anti-neoplastic agents or radiation therapy. Insome embodiments, the second therapeutic agent is selected fromalkylating agents, anti-metabolites, epidophyllotoxins; antineoplasticenzymes, topoisomerase inhibitors, procarbazines, mitoxantrones,platinum coordination complexes, biological response modifiers andgrowth inhibitors, hormonal/anti-hormonal therapeutic agents,hematopoietic growth factors, aromatase inhibitors, anti-estrogens,anti-androgens, corticosteroids, gonadorelin agonists, microtubuleactive agents, nitrosoureas, lipid or protein kinase targeting agents,immunomodulatory drugs (IMiDs), protein or lipid phosphatase targetingagents, anti-angiogenic agents, Akt inhibitors, IGF-I inhibitors, FGF3modulators, mTOR inhibitors, Smac mimetics, HDAC inhibitors, agents thatinduce cell differentiation, bradykinin 1 receptor antagonists,angiotensin II antagonists, cyclooxygenase inhibitors, heparanaseinhibitors, lymphokine inhibitors, cytokine inhibitors, IKK inhibitors,P38MAPK inhibitors, HSP90 inhibitors, multikinase inhibitors,bisphosphonate, rapamycin derivatives, anti-apoptotic pathwayinhibitors, apoptotic pathway agonists, PPAR agonists, RAR agonists,inhibitors of Ras isoforms, telomerase inhibitors, protease inhibitors,metalloproteinase inhibitors, aminopeptidase inhibitors, SHIPactivators - AQX-MN100, Humax-CD20 (ofatumumab), CD20 antagonists,IL2-diptheria toxin fusions, or combinations thereof. In someembodiments, the antimicrobial agent is an antiviral, antibacterial orantifungal agent. Non-limiting exemplary antibacterial agent(s) includethose classified as aminoglycosides, beta lactams, quinolones orfluoroquinolones, macrolides, sulfonamides, sulfamethaxozoles,tetracyclines, streptogramins, oxazolidinones (such as linezolid),clindamycins, lincomycins, rifamycins, glycopeptides, polymxins.lipo-peptide antibiotics, as well as pharmacologically acceptable sodiumsalts, pharmacologically acceptable calcium salts, pharmacologicallyacceptable potassium salts, lipid formulations, derivatives and/oranalogs of the above. Non-limiting exemplary classes of antifungalagents include imidazoles or triazoles such as clotrimazole, miconazole,ketoconazole, econazole, butoconazole, omoconazole, oxiconazole,terconazole, itraconazole, fluconazole, voriconazole (UK 109,496),posaconazole, ravuconazole or flutrimazole; the polyene antifungals suchas amphotericin B, liposomal amphoterecin B, natamycin, nystatin andnystatin lipid formualtions; the cell wall active cyclic lipopeptideantifungals, including the echinocandins such as caspofungin,micafungin, anidulfungin, cilofungin; LY121019; LY303366; the allylaminegroup of antifungals such as terbinafme. Yet other non-limiting examplesof antifungal agents include naftifine, tolnaftate, mediocidin,candicidin, trichomycin, hamycin, aurefungin, ascosin, ayfattin,azacolutin, trichomycin, levorin, heptamycin, candimycin, griseofulvin,BF-796, MTCH 24, BTG-137586, pradimicins (MNS 18184), benanomicin;ambisome; nikkomycin Z; flucytosine, or perimycin. Non-limiting examplesof antiviral agents include cidofovir, amantadine, rimantadine,acyclovir, gancyclovir, pencyclovir, famciclovir, foscamet, ribavirin,or valcyclovir. In some embodiments, the antimicrobial agent is aninnate immune peptide or proteins. Some exemplary classes of innatepeptides or proteins are transferrins, lactoferrins, defensins,phospholipases, lysozyme, cathelicidins, serprocidins, bacteriocidalpermeability increasing proteins, amphipathic alpha helical peptides,and other synthetic antimicrobial proteins. In some embodiments, theantimicrobial agent is an antiseptic agent.

In some embodiments, the second therapeutic agent is selected fromARRY-797, dacarbazine (DTIC), actinomycins C₂, C₃, D, and F₁,cyclophosphamide, melphalan, estramustine, maytansinol, rifamycin,streptovaricin, doxorubicin, daunorubicin, epirubicin, idarubicin,detorubicin, carminomycin, esorubicin, mitoxantrone, bleomycins A, A₂,and B, camptothecin, Irinotecan, Topotecan, 9-aminocamptothecin, 10,11-methylenedioxycamptothecin, 9-nitrocamptothecin, bortezomib,temozolomide, TAS103, NPI0052, combretastatin, combretastatin A-2,combretastatin A-4, calicheamicins, neocarcinostatins, epothilones A B,C, and semi-synthetic variants, Herceptin, Rituxan, CD40 antibodies,asparaginase, interleukins, interferons, leuprolide, and pegaspargase,5-fluorouracil, fluorodeoxyuridine, ptorafur, 5′-deoxyfluorouridine,UFT, MITC, S-1 capecitabine, diethylstilbestrol, tamoxifen, toremefine,tolmudex, thymitaq, flutamide, fluoxymesterone, bicalutamide,finasteride, estradiol, trioxifene, dexamethasone, leuproelin acetate,estramustine, droloxifene, medroxyprogesterone, megesterol acetate,aminoglutethimide, testolactone, testosterone, diethylstilbestrol,hydroxyprogesterone, mitomycins A, B and C, porfiromycin, cisplatin,carboplatin, oxaliplatin, tetraplatin, platinum-DACH, ormaplatin,thalidomide, lenalidomide, CI-973, telomestatin, CHIR258, Rad 001, SAHA,Tubacin, 17-AAG, sorafenib, JM-216, podophyllotoxin, epipodophyllotoxin,etoposide, teniposide, Tarceva, Iressa, Imatinib, Miltefosine,Perifosine, aminopterin, methotrexate, methopterin,dichloro-methotrexate, 6-mercaptopurine, thioguanine, azattuoprine,allopurinol, cladribine, fludarabine, pentostatin, 2-chloroadenosine,deoxycytidine, cytosine arabinoside, cytarabine, azacitidine,5-azacytosine, gencitabine, 5-azacytosine-arabinoside, vincristine,vinblastine, vinorelbine, leurosine, leurosidine and vindesine,paclitaxel, taxotere and/or docetaxel.

In some embodiments, the second active agent is niacin, a fibrate, astatin, a Apo-A1 mimetic polypeptide (e.g., DF-4, Novartis), an apoA-Itranscriptional up-regulator, an ACAT inhibitor, a CETP modulator,Glycoprotein (GP) IIb/IIIa receptor antagonists, P2Y12 receptorantagonists, Lp-PLA2-inhibitors, an anti-tumor necrosis factor (TNF)agent, an interleukin-1 (IL-1) receptor antagonist, an interleukin-2(IL-2) receptor antagonist, an interleukin-6 (IL-6) receptor antagonist,an interleukin-12 (IL-12) receptor antagonist, an interleukin-17 (IL-17)receptor antagonist, an interleukin-23 (IL-23) receptor antagonist, acytotoxic agent, an antimicrobial agent, an immunomodulatory agent, anantibiotic, a T-cell co-stimulatory blocker, a disorder-modifyinganti-rheumatic agent, a B cell depleting agent, an immunosuppressiveagent, an anti-lymphocyte antibody, an alkylating agent, ananti-metabolite, a plant alkaloid, a terpenoids, a topoisomeraseinhibitor, an anti-tumor antibiotic, a monoclonal antibody, a hormonaltherapy (e.g., aromatase inhibitors), or combinations thereof.

In some embodiments, the second active agent is an anti-TGF-β antibody,an anti-TGF-β receptor blocking antibody, an anti-TNF antibody, ananti-TNF receptor blocking antibody, an anti-IL1β antibody, an anti-IL1βreceptor blocking antibody, an anti-IL-2 antibody, an anti-IL-2 receptorblocking antibody, an anti-IL-6 antibody, an anti-IL-6 receptor blockingantibody, an anti IL-12 antibody, an anti IL-12 receptor blockingantibody, an anti-IL-17 antibody, anti-IL-17 receptor blocking antibody,an anti-IL-23 antibody, or an anti-IL-23 receptor blocking antibody.

In some embodiments, the second active agent is niacin, bezafibrate;ciprofibrate; clofibrate; gemfibrozil; fenofibrate; DF4(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2); DF5; RVX-208(Resverlogix); avasimibe; pactimibe sulfate (CS-505); CI-1011(2,6-diisopropylphenyl [(2, 4,6-triisopropylphenyl)acetyl]sulfamate);CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); VULM1457(1-(2,6-diisopropyl-phenyl)-3-[4-(4′-nitrophenylthio)phenyl] urea);CI-976 (2,2-dimethyl-N-(2,4,6- trimethoxyphenyl)dodecanamide); E-5324(n-butyl-N′-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea);HL-004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide); KY-455(N-(4,6- dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide); FY-087(N-[2-[N′-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide);MCC-147 (Mitsubishi Pharma); F 12511((S)-2′,3′,5′-trimethyl-4′-hydroxy-alpha-dodecylthioacetanilide);SMP-500 (Sumitomo Pharmaceuticals); CL 277082(2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-(hepthyl)urea);F-1394((1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate); CP-113818(N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acidamide); YM-750; torcetrapib; anacetrapid; JTT-705 (Japan Tobacco/Roche);abciximab; eptifibatide; tirofiban; roxifiban; variabilin; XV 459(N(3)-(2-(3-(4-formamidinophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butyloxycarbonyl)-2,3-diaminopropionate);SR 121566A (3-[N-{4-[4-(aminoiminomethyl)phenyl ]-1,3-thiazol-2-yl}-N-(1-carboxymethylpiperid-4-yl) aminol propionic acid, trihydrochloride);FK419 ((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl]piperidin-3-ylcarbonyl] amino] propionic acid trihydrate); clopidogrel;prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS 2395(2,2-Dimethyl-propionic acid 3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl ester); BX 667 (BerlexBiosciences); BX 048 (Berlex Biosciences); darapladib (SB 480848);SB-435495 (GlaxoSmithKline); SB-222657 (GlaxoSmithKline); SB-253514(GlaxoSmithKline); alefacept, efalizumab, methotrexate, acitretin,isotretinoin, hydroxyurea, mycophenolate mofetil, sulfasalazine,6-Thioguanine, Dovonex, Taclonex, betamethasone, tazarotene,hydroxychloroquine, sulfasalazine, etanercept, adalimumab, infliximab,abatacept, rituximab, trastuzumab, anti-CD45 monoclonal antibody AHN-12(NCI), Iodine-131 Anti-B1 Antibody (Corixa Corp.), anti-CD66 monoclonalantibody BW 250/183 (NCI, Southampton General Hospital), anti-CD45monoclonal antibody (NCI, Baylor College of Medicine), antibodyanti-anb3 integrin (NCI), BIW-8962 (BioWa Inc.), antibody BC8 (NCI),antibody muJ591 (NCI), indium In 111 monoclonal antibody MN-14 (NCI),yttrium Y 90 monoclonal antibody MN-14 (NCI), F105 Monoclonal Antibody(NIAID), Monoclonal Antibody RAV12 (Raven Biotechnologies), CAT-192(Human Anti-TGF-Beta1 Monoclonal Antibody, Genzyme), antibody 3F8 (NCI),177Lu-J591 (Weill Medical College of Cornell University), TB-403(BioInvent International AB), anakinra, azathioprine, cyclophosphamide,cyclosporine A, leflunomide, d-penicillamine, amitriptyline, ornortriptyline, chlorambucil, nitrogen mustard, prasterone, LJP 394(abetimus sodium), LJP 1082 (La Jolla Pharmaceutical), eculizumab,belibumab, rhuCD40L (NIAID), epratuzumab, sirolimus, tacrolimus,pimecrolimus, thalidomide, antithymocyte globulin-equine (Atgam,Pharmacia Upjohn), antithymocyte globulin-rabbit (Thymoglobulin,Genzyme), Muromonab-CD3 (FDA Office of Orphan Products Development),basiliximab, daclizumab, riluzole, cladribine, natalizumab, interferonbeta-1b, interferon beta-1a, tizanidine, baclofen, mesalazine, asacol,pentasa, mesalamine, balsalazide, olsalazine, 6-mercaptopurine, AIN457(Anti IL-17 Monoclonal Antibody, Novartis), theophylline, D2E7 (a humananti-TNF mAb from Knoll Pharmaceuticals), Mepolizumab (Anti-IL-5antibody, SB 240563), Canakinumab (Anti-IL-1 Beta Antibody, NIAMS),Anti-IL-2 Receptor Antibody (Daclizumab, NHLBI), CNTO 328 (Anti IL-6Monoclonal Antibody, Centocor), ACZ885 (fully humananti-interleukin-1beta monoclonal antibody, Novartis), CNTO 1275 (FullyHuman Anti-IL-12 Monoclonal Antibody, Centocor),(3S)-N-hydroxy-4-({4-[(4-hydroxy-2-butynyl)oxy]phenyl}sulfonyl)-2,2-dimet-hyl-3-thiomorpholine carboxamide (apratastat), golimumab (CNTO 148),Onercept, BG9924 (Biogen Idec), Certolizumab Pegol (CDP870, UCB Pharma),AZD9056 (AstraZeneca), AZD5069 (AstraZeneca), AZD9668 (AstraZeneca),AZD7928 (AstraZeneca), AZD2914 (AstraZeneca), AZD6067 (AstraZeneca),AZD3342 (AstraZeneca), AZD8309 (AstraZeneca), ),[(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronicacid (Bortezomib), AMG-714, (Anti-IL 15 Human Monoclonal Antibody,Amgen), ABT-874 (Anti IL-12 monoclonal antibody, Abbott Labs),MRA(Tocilizumab, an Anti IL-6 Receptor Monoclonal Antibody, ChugaiPharmaceutical), CAT-354 (a human anti-interleukin-13 monoclonalantibody, Cambridge Antibody Technology, MedImmune), aspirin, salicylicacid, gentisic acid, choline magnesium salicylate, choline salicylate,choline magnesium salicylate, choline salicylate, magnesium salicylate,sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofencalcium, flurobiprofen, ibuprofen, ketoprofen, nabutone, ketolorac,ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac,indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium,mefenamic acid, piroxicam, meloxicam, celecoxib, rofecoxib, valdecoxib,parecoxib, etoricoxib, lumiracoxib, CS-502 (Sankyo), JTE-522 (JapanTobacco Inc.), L-745,337 (Almirall), NS398 (Sigma), betamethasone(Celestone), prednisone (Deltasone), alclometasone, aldosterone,amcinonide, beclometasone, betamethasone, budesonide, ciclesonide,clobetasol, clobetasone, clocortolone, cloprednol, cortisone,cortivazol, deflazacort, deoxycorticosterone, desonide, desoximetasone,desoxycortone, dexamethasone, diflorasone, diflucortolone,difluprednate, fluclorolone, fludrocortisone, fludroxycortide,flumetasone, flunisolide, fluocinolone acetonide, fluocinonide,fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene,fluticasone, formocortal, formoterol, halcinonide, halometasone,hydrocortisone, hydrocortisone aceponate, hydrocortisone buteprate,hydrocortisone butyrate, loteprednol, medrysone, meprednisone,methylprednisolone, methylprednisolone aceponate, mometasone furoate,paramethasone, prednicarbate, prednisone, rimexolone, tixocortol,triamcinolone, ulobetasol; cisplatin; carboplatin; oxaliplatin;mechlorethamine; cyclophosphamide; chlorambucil; vincristine;vinblastine; vinorelbine; vindesine; azathioprine; mercaptopurine;fludarabine; pentostatin; cladribine; 5-fluorouracil (5FU); floxuridine(FUDR); cytosine arabinoside; methotrexate; trimethoprim; pyrimethamine;pemetrexed; paclitaxel; docetaxel; etoposide; teniposide; irinotecan;topotecan; amsacrine; etoposide; etoposide phosphate; teniposide;dactinomycin; doxorubicin; daunorubicin; valrubicine; idarubicine;epirubicin; bleomycin; plicamycin; mitomycin; trastuzumab; cetuximab;rituximab; bevacizumab; finasteride; goserelin; aminoglutethimide;anastrozole; letrozole; vorozole; exemestane; 4-androstene-3,6,17-trione(“6-OXO”; 1,4,6-androstatrien-3,17-dione (ATD); formestane;testolactone; fadrozole; or combinations thereof.

In some embodiments, the second therapeutic agent is an antibiotic. Insome embodiments, the second therapeutic agent is an anti-bacterialagent. In some embodiments, the second therapeutic agent is amikacin,gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramycin,paromomycin, geldanmycin, herbimycin, loracarbef, ertapenem, doripenem,imipenem, cilastatin, meropenem, cefadroxil, cefazolin, cefalotin,cefalexin, cefaclor, cefamandole, cefoxitin, defprozil, cefuroxime,cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime,ceftazidime, ceftibuten, ceftizoxime, ceftriaxone, cefepime,ceftobiprole, teicoplanin, vancomycin, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, troleandomycin,telithromycin, spectinomycin, aztreonam, amoxicillin, ampicillin,azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin,mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin,ticarcillan, bacitracin, colistin, polymyxin B, ciprofloxacin, enoxacin,gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin,ofloxacin, trovfloxacin, mafenide, prontosil, sulfacetamide,sulfamethizole, sulfanimilimde, sulfsalazine, sulfsioxazole,trimethoprim, demeclocycline, doxycycline, minocycline, oxtetracycline,tetracycline, arsphenamine, chloramphenicol, clindamycin, lincomycin,ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid,linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin,pyrazinamide, quinuspristin/dalfopristin, rifampin, tinidazole, andcombinations thereof.

In some embodiments, the second therapeutic agent is an anti-viralagent. In some embodiments, the second therapeutic agent is acyclovir,famciclovir, valacyclovir, abacavir, aciclovir, adfovir, amantadine,amprenavir, arbidol., atazanavir, artipla, brivudine, cidofovir,combivir, edoxudine, efavirenz, emtricitabine, enfuvirtide, entecavir,fomvirsen, fosamprenavir, foscarnet, fosfonet, ganciclovir, gardasil,ibacitabine, imunovir, idoxuridine, imiquimod, indinavir, inosine,integrase inhibitors, interferons, including interferon type I (e.g. IFNα and IFN β), interferon type II, interferon type III, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, tenofovir, tenofovir disoproxil, tipranavir, trifluridine,trizivir, tromantadine, truvada, valganciclovir, vicriviroc, vidarabine,viramidine, zalcitabine, zanamivir, zidovudine, and combinationsthereof.

In some embodiments, the second therapeutic agent is an anti-fungalagent. In some embodiments, the second therapeutic agent is amrolfine,utenafine, naftifine, terbinafine, flucytosine, fluconazole,itraconazole, ketoconazole, posaconazole, ravuconazole, voriconazole,clotrimazole, econazole, miconazole, oxiconazole, sulconazole,terconazole, tioconazole, nikkomycin Z, caspofungin, micafungin,anidulafungin, amphotericin B, liposomal nystastin, pimaricin,griseofulvin, ciclopirox olamine, haloprogin, tolnaftate, undecylenate,clioquinol, and combinations thereof.

In some embodiments, the second therapeutic agent is an anti-parasiticagent. In some embodiments, the second therapeutic agent is amitraz,amoscanate, avermectin, carbadox, diethylcarbamizine, dimetridazole,diminazene, ivermectin, macrofilaricide, malathion, mitaban,oxamniquine, permethrin, praziquantel, prantel pamoate, selamectin,sodium stibogluconate, thiabendazole, and combinations thereof.

Combinations With Cells and Tissues

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is co-administered with a cell, a plurality of cells or a tissue.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is co-administered with a therapeutic cell. In some embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein isco-administered with a tissue transplant. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is co-administeredwith a stem cell transplant. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is co-administered with an organtransplant.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) with a tissuetransplant. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein is administered before or after a tissue transplant. Insome embodiments, the time period between administration of annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and the tissuetransplant ranges from a few minutes to several hours, depending uponthe properties of each pharmaceutical agent, such as potency,solubility, bioavailability, plasma half-life and kinetic profile of thepharmaceutical agent. In some embodiments, circadian variation of thetarget molecule concentration determines the optimal dose interval. Insome embodiments, the timing between the administration of annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein and a second activeagent is about less than an hour, less than a day, less than a week, orless than a month.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is co-administered with a tissue transplant and animmunosuppressive agent. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein is co-administered with a tissuetransplant and a calcineurin inhibitor (e.g., cyclosporin ortacrolimus); an mTOR inhibitor (sirolimus; everolimus); ananti-proliferative agent (azathioprine or mycophenolic acid); acorticosteroid (e.g., prednisolone or hydrocortisone); a monoclonalanti-IL-2Rα receptor antibody (e.g., basiliximab or daclizumab); apolyclonal anti-T-cell antibodies (e.g., anti-thymocyte globulin (ATG)or anti-lymphocyte globulin (ALG)); or combinations thereof.

In some embodiments, a tissue is coated with an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein. In some embodiments, a pluralityof stem cells are coated with an nHC-HA/PTX3 or rcHC-HA/PTX3 complexdisclosed herein. In some embodiments, an organ is coated with annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein. In someembodiments, coating a tissue with an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein prevent a tissue from being acted upon by thehost immune system.

In some embodiments, an organ, tissue, or plurality of stem cells iscontacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein.In some embodiments, an organ, tissue, or plurality of stem cells iscontacted with a composition comprising an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein. In some embodiments, the composition has a pHof about 7.0 to about 7.5. In some embodiments, the composition has a pHof 7.4. In some embodiments, the composition further comprisespotassium, magnesium, and raffinose. In some embodiments, thecomposition further comprises at least one of adenosine, glutathione,allopurinol, and hydroxyethyl starch. In some embodiments, thecomposition is UW solution supplemented with an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein.

In some embodiments, the organ, tissue, or plurality of stem cells arecontacted with an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed hereinfor about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about4 hours, about 5 hours, about 6 hours, about 12 hours, about 24 hours,about 36 hours, or about 48 hours. In some embodiments, the contactingoccurs at a temperature that protects tissues and vascular conditioning(e.g., less than ambient temperature). In some embodiments, thecontacting occurs at 4° C.

Medical Device Combinations

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosedherein is co-administered with a medical device. In some embodiments,medical device or a portion thereof is contacted with an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex disclosed herein is use to coat amedical device or a portion thereof as described elsewhere herein. Insome embodiment, administration of an nHC-HA/PTX3 or rcHC-HA/PTX3complex disclosed herein in combination with a medical device reduces,inhibits or prevents inflammatory reactions against the implantedmedical device. In some embodiment, administration of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex disclosed herein in combination with a medicaldevice reduces, inhibits or prevents the formation of infectiousbiofilms that are produce by microorganism growth on the implantedmedical device (i.e. chronic biofilm infection). Exemplary of suchbiofilms are those produce by bacteria, such as but not limited to,Staphylococcus aureus.

Articles of Manufacture and Kits

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. Examples of pharmaceuticalpackaging materials include, but are not limited to, blister packs,bottles, tubes, inhalers, inhalers (e.g., pressurized metered doseinhalers (MDI), dry powder inhalers (DPI), nebulizers (e.g., jet orultrasonic nebulizers) and other single breath liquid systems), pumps,bags, vials, containers, syringes, bottles, and any packaging materialsuitable for a selected formulation and intended mode of administrationand treatment. In some embodiments, the pharmaceutical composition isincorporated in, applied to or coated on a medical devices, such asimplants, catheters, artificial joints, stents, valves, nanoparticles,or microcapsules.

In some embodiments, the pharmaceutical compositions or combinationsprovided herein are provided as kits. Kits optionally include one ormore components such as instructions for use, devices and additionalreagents (e.g., sterilized water or saline solutions for dilution of thecompositions and/or reconstitution of lyophilized protein), andcomponents, such as tubes, containers and syringes for practice of themethods. Exemplary kits include the pharmaceutical compositions orcombinations provided herein, and optionally include instructions foruse, a device for administering the pharmaceutical compositions orcombinations to a subject, a device for detecting the nHC-HA/PTX3 orrcHC-HA/PTX3 complexes in a subject, a device for detecting thenHC-HA/PTX3 or rcHC-HA/PTX3 complexes in samples obtained from asubject, and a device for administering an additional therapeutic agentto a subject.

The kit can, optionally, include instructions. Instructions typicallyinclude a tangible expression describing the nHC-HA/PTX3 or rcHC-HA/PTX3complexes and, optionally, other components included in the kit, andmethods for administration, including methods for determining the properstate of the subject, the proper dosage amount, dosing regimens, and theproper administration method for administering the nHC-HA/PTX3 orrcHC-HA/PTX3 complexes. In some embodiments, instructions includeguidance for monitoring the subject over the duration of the treatmenttime

In some embodiments, kits include a pharmaceutical composition describedherein and an item for diagnosis. For example, such kits include an itemfor measuring the concentration, amount or activity of the selectednHC-HA/PTX3 or rcHC-HA/PTX3 complexes in a subject.

In some embodiments, kits provided herein include a device foradministering the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes to a subject. Insome embodiments, any of a variety of devices known in the art foradministering medications to a subject -is included in the kits providedherein. Exemplary devices include, but are not limited to, an inhaler(e.g., pressurized metered dose inhaler (MDI), dry powder inhaler (DPI),nebulizer (e.g., jet or ultrasonic nebulizers) and other single breathliquid system), a hypodermic needle, an intravenous needle, a catheter,and a liquid dispenser such as an eyedropper. Typically the device foradministering the nHC-HA/PTX3 or rcHC-HA/PTX3 complexes of the kit willbe compatible with the desired method of administration of thenHC-HA/PTX3 or rcHC-HA/PTX3 complexes.

Expansion of Stem Cell Cultures

Disclosed herein, in certain embodiments, are methods of expanding anisolated stem cell on a substrate that comprises an nHC-HA/PTX3 orrcHC-HA/PTX3 complex provided herein. As described herein, HC-HA/PTX3complexes promote the aggregation of stem cells, prevent differentiationof the cells and preserves expression of stem cell markers.

In some embodiments, the expansion on an nHC-HA/PTX3 complex orrcHC-HA/PTX3 complex preserves expression of one or more of embryonicstem cell (ESC) markers (e.g. Oct4, Nanog, Sox2 (SRY (sex determiningregion Y)-box 2), Rex1 (Zfp42), SSEA4 (stage-specific embryonicantigen-4), MYC/c-Myc and KLF4, pericyte markers (e.g. NG2 (neuron-glialantigen 2/Chondroitin sulfate proteoglycan 4(CSPG4)), PDGFR- β(Platelet-derived growth factor receptor B), and α-SMA (α-smooth muscleactin)), and angiogenic markers (e.g. CD133/2, FLK-1 (VEGF-R2, Ly-73),vWF (von Willebrand factor), CD34, CD31 (PECAM-1) and CD146). In someembodiments, the expression of the stem cell marker is determined byconventional methods, such as for example, protein expression analysis(e.g. Western blotting, immunofluorescence, immunohistochemistry,fluorescence activated cell sorting) or mRNA analysis (e.g. polymerasechain reaction (PCR) or Northern).

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 suppresses TGF- βsignaling in a cultured cell. In some embodiments, nHC-HA/PTX3 orrcHC-HA/PTX3 suppresses TGF- β signaling in a cultured stem cell. Insome embodiments, suppression of TGF- β signaling refers to a decreasein the activity or expression of one or more proteins or markers in theTGF- β cell signaling pathway such as pSMAD ⅔ signaling, α smooth muscleformation, in a cell in the presence of the nHC-HA/PTX3 or rcHC-HA/PTX3complex compared to the absence of the nHC-HA/PTX3 or rcHC-HA/PTX3complex.

In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3 induces BMP signalingin a cultured cell. In some embodiments, nHC-HA/PTX3 or rcHC-HA/PTX3induces BMP signaling in a cultured stem cell. In some embodiments,suppression of TGF- β signaling refers to an increase in the activity orexpression of one or more proteins in the BMP signaling pathway, such asBMP-2, BMP-4, BMP-6 and pSMAD1/5/8, in a cell in the presence of thenHC-HA/PTX3 or rcHC-HA/PTX3 complex compared to the absence of thenHC-HA/PTX3 or rcHC-HA/PTX3 complex.

In some embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is an embryonic stem cell. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is an adultstem cell. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3is a fetal stem cell. In some embodiments,the isolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is aninduced pluripotent/progenitor stem cell (iPSC).

In some embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3is a mesenchymal stem cell. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is an adiposestem cell (ASC). In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3is an umbilical cord stem cell. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3is an amniotic membrane stem cell. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is a limbalcell, such as a limbal niche cell or a limbal epithelial progenitorcell. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3is an endothelial stem cell. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is a hematopoietic stem cell. In some embodiments, theisolated stem cell is a bone marrow stem cell. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is a neuralstem cell. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3is an endothelial progenitor cell. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is a skeletal muscle stem cell. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is a mammarystem cell. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3 is an intestinal stem cell.

In some embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is an induced pluripotent/progenitor stem cell (iPSC). Insome embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is an induced pluripotent stem cell derived from an adultdifferentiated or partially differentiated cell. In some embodiments,the isolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is aninduced pluripotent stem cell derived from a fibroblast. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is an induced pluripotent stem cell derived from a humancorneal fibroblast.

In some embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from a fetal tissue, such as placental tissue oran umbilical cord tissue. In some embodiments, the isolated stem cellcultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derived from amnioticmembrane. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3 is derived from adipose tissue. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from limbal tissue. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derivedfrom bone marrow. In some embodiments, the isolated stem cell culturedon nHC-HA/PTX3 or rcHC-HA/PTX3 is derived from endothelial tissue. Insome embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from limbal tissue. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derivedfrom neural tissue, In some embodiments, the isolated stem cell culturedon nHC-HA/PTX3 or rcHC-HA/PTX3 is derived from limbal tissue. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from skeletal muscle. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derivedfrom the skin. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3 is derived from the digestive system. Insome embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from the pancreas. In some embodiments, theisolated stem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derivedfrom the liver. In some embodiments, the isolated stem cell cultured onnHC-HA/PTX3 or rcHC-HA/PTX3 is derived from the olfactory mucosa. Insome embodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from a germ cell population. In someembodiments, the isolated stem cell cultured on nHC-HA/PTX3 orrcHC-HA/PTX3 is derived from blood. In some embodiments, the isolatedstem cell cultured on nHC-HA/PTX3 or rcHC-HA/PTX3 is derived fromumbilical cord blood.

In some embodiments, the HC-HA/PTX3 complex is an nHC-HA/PTX3 isolatedfrom amniotic membrane or umbilical cord. In some embodiments, theHC-HA/PTX3 complex is a reconstituted HC-HA complex. In someembodiments, HA is covalently linked to HC. In some embodiments, the HCof IαI is heavy chain 1 (HC1). In some embodiments, the HC-HA complexcomprises pentraxin 3 (PTX3).

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises asmall leucine rich proteoglycan (SLRP). In some embodiments, thenHC-HA/PTX3 or rcHC-HA/PTX3 complex comprises a class I, class II orclass II SLRP. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complex comprises PTX3 and a small leucine rich proteoglycan (SLRP). Insome embodiments, the small leucine-rich proteoglycan is selected fromamong class I SLRPs, such as decorin and biglycan. In some embodiments,the small leucine-rich proteoglycan is selected from among class IISLRPs, such as fibromodulin, lumican, PRELP (proline arginine rich endleucine-rich protein), keratocan, and osteoadherin. In some embodiments,the small leucine-rich proteoglycan is selected from among class IIISLRPs, such as epipycan and osteoglycin.

In some embodiments, the isolated stem cell is expanded on a substratecomprising immobilized nHC-HA/PTX3 or rcHC-HA/PTX3. In some embodiments,the immobilized nHC-HA/PTX3 or rcHC-HA/PTX3comprises one or more smallleucine rich proteoglycans (SLRPs). In some embodiments, the SLRP isselected from among bikunin, decorin, biglycan, and osteoadherin. Insome embodiments, the small leucine-rich protein comprises aglycosaminoglycan. In some embodiments, the small leucine-richproteoglycan comprises keratan sulfate.

In some embodiments, the isolated stem cell is expanded in a culturemedium comprising nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In someembodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3comprises one or more smallleucine rich proteoglycans (SLRPs). In some embodiments, the SLRP isselected from among bikunin, decorin, biglycan, and osteoadherin. Insome embodiments, the small leucine-rich protein comprises aglycosaminoglycan. In some embodiments, the small leucine-richproteoglycan comprises keratan sulfate. In some embodiments, the mediumis embryonic stem cell medium, modified embryonic stem cell medium,supplemented hormonal epithelial medium, and/or a combination thereof.In some embodiments, the medium is supplemented with one or more growthfactors. In some embodiments, the medium is supplemented with EGF, bFGFand/or LIF. In some embodiments, the medium is supplemented with aninhibitor of Rho-associated kinase (ROCK inhibitor).

Inducing and Maintaining Pluripotency

Disclosed herein, in certain embodiments, are methods of inducingpluripotency in a cell or maintaining pluripotency of a stem cell on asubstrate that comprises an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. Asdescribed herein, HC-HA/PTX3 complexes assist in the maintenance of stemcell marker expression and prevent differentiation of the cells oversuccessive passages of a stem cell population. In addition, as describedherein, HC-HA/PTX3 complexes promote the induction of stem cellproperties in a differentiated or partially differentiated population ofcells.

In certain embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex promotesor induces pluripotency of a differentiated or partially differentiatedcell. In certain embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexpromotes or induces pluripotency of a differentiated or partiallydifferentiated cell compared to a differentiated or partiallydifferentiated cell cultured in the absence of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In an exemplary method, a differentiated cell orpartially differentiated cell is cultured on a substrate comprisingnHC-HA/PTX3 or rcHC-HA/PTX3 complex, whereby pluripotency is induced inthe cell.

In certain embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex furtherpromotes or induces pluripotency of a stem cell. In certain embodiments,an nHC-HA/PTX3 or rcHC-HA/PTX3 complex further promotes or inducespluripotency of a stem cell compared to a stem cultured in the absenceof an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In an exemplary method, astem cell is cultured on a substrate comprising nHC-HA/PTX3 orrcHC-HA/PTX3 complex, whereby pluripotency is maintained in the stemcell. In an exemplary method, a stem cell is cultured on a substratecomprising nHC-HA/PTX3 or rcHC-HA/PTX3 complex, whereby pluripotency isfurther induced in the stem cell.

Using genetic reprogramming with protein transcription factors,pluripotent stem cells equivalent to embryonic stem cells have beenderived from human adult skin tissue. iPSC cells are typically derivedby transfection of certain stem cell-associated genes intonon-pluripotent cells, such as adult fibroblasts. Transfection istypically achieved through viral vectors, such as retroviruses, wherethe pluripotency gene is operably linked to a promoter for geneexpression. Four key pluripotency genes essential for the production ofpluripotent stem cells are Oct-¾ (Pou5f1), Sox2, c-Myc, and Klf4. Othergenes can enhance the efficiency of induction. In some studies, Oct4,Sox2, Nanog, and Lin28 have been employed to induce pluripotency. Incertain instances, after 3-4 weeks, small numbers of transfected cellsbegin to become morphologically and biochemically similar to pluripotentstem cells, and are typically isolated through morphological selection,doubling time, or through a reporter gene and antibiotic selection.

In some embodiments, methods are provided for inducing pluripotency in adifferentiated or partially differentiated cell using heterologousexpression of fewer than four of the essential transcription factorsOct-¾ (Pou5f1), Sox2, c-Myc, and Klf4. In some embodiments, a method forinducing pluripotency is provided where use of an nHC-HA/PTX3 orrcHC-HA/PTX3 complex enhances the induction of pluripotency of adifferentiated or partially differentiated cell that expresses at leastone of Oct-¾ (Pou5f1), Sox2, c-Myc, and/or Klf4 by heterologous genetransfer. In some embodiments, a method for inducing pluripotency isprovided where use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhancesthe induction of pluripotency of a differentiated or partiallydifferentiated cell that expresses one, two or three factors selectedfrom among Oct-¾ (Pou5f1), Sox2, c-Myc, and/or Klf4 by heterologous genetransfer. In some embodiments, a method for inducing pluripotency isprovided where use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhancesthe induction of pluripotency of a differentiated or partiallydifferentiated cell that expresses Oct-¾ (Pou5f1) by heterologous genetransfer. In some embodiments, a method for inducing pluripotency isprovided where use of an nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhancesthe induction of pluripotency of a differentiated or partiallydifferentiated cell that Sox2 by heterologous gene transfer. In someembodiments, a method for inducing pluripotency is provided where use ofan nHC-HA/PTX3 or rcHC-HA/PTX3 complex enhances the induction ofpluripotency of a differentiated or partially differentiated cell thatexpresses c-Myc by heterologous gene transfer. In some embodiments, amethod for inducing pluripotency is provided where use of an nHC-HA/PTX3or rcHC-HA/PTX3 complex enhances the induction of pluripotency of adifferentiated or partially differentiated cell that expresses Klf4 byheterologous gene transfer.

In some embodiments, a differentiated or partially differentiated cellis transduced to express one or more of Oct-¾ (Pou5f1), SOX2, c-Myc, andKlf4; and the transduced cell is cultured on a substrate comprising annHC-HA/PTX3 or rcHC-HA/PTX3 complex. In some embodiments, adifferentiated or partially differentiated cell is transduced to expressat least one of Oct-¾ (Pou5f1), SOX2, c-Myc, and Klf4; and thetransduced cell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, a differentiated or partiallydifferentiated cell is transduced to express one, two or three of Oct-¾(Pou5f1), SOX2, c-Myc, and Klf4; and the transduced cell is cultured ona substrate comprising an nHC-HA/PTX3 or rcHC-HA/PTX3 complex. In someembodiments, a differentiated or partially differentiated cell istransduced to express Oct-¾ (Pou5f1), SOX2, c-Myc, and Klf4; and thetransduced cell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, a differentiated or partiallydifferentiated cell is transduced to express Oct-¾ (Pou5f1); and thetransduced cell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, a differentiated or partiallydifferentiated cell is transduced to express SOX2; and the transducedcell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, a differentiated or partiallydifferentiated cell is transduced to express c-Myc; and the transducedcell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, a differentiated or partiallydifferentiated cell is transduced to express Klf4; and the transducedcell is cultured on a substrate comprising an nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, the cell is transduced toexpress one or more additional genes, such as for example, Nanog, Fbx15,ERas, ECAT15-2, Tcl1, and β-catenin.

In some embodiments, a differentiated or partially differentiated cellis transduced with a viral vector containing one or more genes encodingone or more of Oct-¾ (Pou5f1), SOX2, c-Myc, and Klf4. In someembodiments, a differentiated or partially differentiated cell istransduced with two or more viral vectors containing one or more genesencoding one or more of Oct-¾ (Pou5f1), SOX2, c-Myc, and Klf4.

Various methods for the induction, culturing and maintainance of inducedpluripotent stem cells and assessment of the pluripotency of inducedstem cells, including assessment of stem cell markers and induction ofdifferent cell lineages, are well known in the art and include, forexample, methods described in U.S. Pat. Nos. 7,682,828, 8,048,999,8,211,697, 7,951,592, and U.S. Pat. Pubs. 2009/0191159 and 2010/000375.

In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complex reduces totime of induction of pluripotency in the transduced cell compared to atransduced cell cultured in the absence of nHC-HA/PTX3 or rcHC-HA/PTX3complex. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3 complexincreases the percentage of transduced cells that are induced topluripotency in a population of transduced cells compared to transducedcells cultured in the absence of nHC-HA/PTX3 or rcHC-HA/PTX3 complexcompared to a transduced cell cultured in the absence of nHC-HA/PTX3 orrcHC-HA/PTX3 complex. In some embodiments, the nHC-HA/PTX3 orrcHC-HA/PTX3 complex enhances the level of pluripotency in thetransduced cell. In some embodiments, the nHC-HA/PTX3 or rcHC-HA/PTX3complex decreases the number of heterologous transcription factorsrequired for induction of pluripotency in the transduced cell.

In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complex providedherein inhibits TGFβ1 signaling in a differentiated cell, a stem cell,or an iPSC. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexprovided herein inhibits nuclear translocation of SMAD2 or SMAD3 in adifferentiated cell, a stem cell, or an iPSC. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein inhibits alphasmooth muscle actin formation in a differentiated cell, a stem cell, oran iPSC. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexprovided herein activates BMP4 signaling in a differentiated cell, astem cell, or an iPSC. In some embodiments, an nHC-HA/PTX3 orrcHC-HA/PTX3 complex provided herein activates BMP6 signaling in adifferentiated cell, a stem cell, or an iPSC. In some embodiments, annHC-HA/PTX3 or rcHC-HA/PTX3 complex provided herein induces expressionof an embryonic cell marker in a differentiated cell, a stem cell, or aniPSC. In some embodiments, an nHC-HA/PTX3 or rcHC-HA/PTX3 complexprovided herein induces expression of c-myc, KLF-4, Nanog, nestin, Oct4,Rex-1, Sox-2, and SSEA-4 in a differentiated cell, a stem cell, or aniPSC.

EXAMPLES

The following examples are included for illustrative purposes only andare not intended to limit the scope of the claimed subject matter.

Example 1. Purification of Native HC-HA/PTX3 (nHC-HA/PTX3) ComplexesFrom Human Amniotic Membrane Extracts (AME) Preparation of AmnioticMembrane Extract (AME) and AM Powder (AMP)

Frozen human AM obtained from Bio-tissue (Miami, FL) was washed 2-3times with PBS to remove the storage medium. To prepare AME, AM wastransferred to a sterile 50 ml centrifuge tube and centrifuged at 4° C.for 5 min at 5000 × g to remove the excess fluid. AM was weighed (~10mg/cm²), transferred to a 100 mm or 150 mm sterile Petri dish, andfrozen in the air phase of a liquid nitrogen container for 20 min beforebeing sliced into small pieces with a disposable scalpel and homogenizedwith Tissue-Tearor (Biospec Products, Inc., Bartlesville, OK) in PBS.The homogenate was mixed at 4° C. for 30 min and centrifuged at 48,000 ×g for 30 min. The supernatant was collected, designated as AME, and usedfor nHC-HA/PTX3 purification or stored at -80° C.

To prepare lyophilized AM powder (AMP), AM frozen in a -80° C. freezerwas transferred to and lyophilized in a bench top lyophilizer (Freezone4.5, Labconco, Kansas City, MO) for 16 hours. The lyophilized AM wasthen micronized into its matrix form (AMP) by a Mixer Mill (Retsch,Newtown, PA). AMP was stored at below -20° C. for further analyses.

Purification of Native HC-HA/PTX3 (nHC-HA/PTX3) Complex

AME was dissolved in CsCl/4M guanidine HCl mixture at an initial densityof 1.35 g/ml, and centrifuged at 125,000 × g for 48 h at 15° C. A totalof 15 fractions (0.8 ml/fraction) were collected from the top to thebottom of each tube. Total protein concentration for each fraction wasdetermined by BCA Protein Assay Kit. Hyaluronan (HA) concentration foreach fraction was determined by an ELISA-based HA Quantitative Test Kitfrom Corgenix (Westminster, CO) (FIG. 1A). Fractions #8-15, whichcontain HA but no detectable proteins, were pooled, and used for asecond ultracentrifugation. A sample of the pooled fractions (designatedAM 1st) was saved for analysis. The pooled fractions were adjusted withCsCl/4M guanidine HCl at an initial density of 1.40 g/ml, centrifuged,and fractionated in the same manner as described above (FIG. 1B).Fractions #3-15, which contained HA but no detectable proteins, werepooled (designated AM 2nd) and dialyzed against distilled water toremove CsCl and guanidine HCl. The dialysate was lyophilized the samemanner as for AMP describe above. Alternatively, the dialysate was mixedwith 3 volumes of 95% (v/v) ethanol containing 1.3% (w/v) potassiumacetate at 0° C. for 1 h. After centrifugation at 15,000 × g, the pelletwas washed with 70% (v/v) ethanol and centrifuged again. The pellet wasbriefly dried by air, stored at -80° C. The powder and pellet weredesignated as nHC-HA/PTX3 complex.

In some instances, the pooled sample went through three or four times ofultracentrifugation. In these ultracentrifugations, only fractions #7-12were pooled and the initial density of CsCl/4M guanidine HCl is at 1.42g/ml. After the third or fourth ultracentrifugation, the pooledfractions #7-12 is designated nHC-HA/PTX3 (3^(rd)) or nHC-HA/PTX3(4^(th)).

AME pooled fractions after 1^(st), 2^(nd), 3^(rd), or 4^(th)ultracentrifugations were treated with or without 0.05 N NaOH at 25° C.for 1 h. Pooled fractions from 1^(st), 2^(nd), 3^(rd), or 4^(th)ultracentrifugation also were digested with 20 units/ml Hyaluronidase(HAase) (Seikagaku Biobusiness Corporation, Tokyo, Japan) at 60° C. for2 h.

Samples from the pooled fractions and the NaOH and HAase treated sampleswere then run on 0.5% agarose gels and analyzed by staining withStains-all dye (FIG. 1C) or by Western blot using antibodies against IαIheavy chain 1 (HC1) (FIGS. 1D and 1F), pentraxin 3 (PTX3) (FIGS. 1E and1G), IαI heavy chain 2 (HC2) (FIG. 1H), IαI heavy chain 3 (HC3) (FIG.1I), bikunin (FIG. 1J), TNF-stimulated gene 6 (TSG-6) (FIG. 1K),thrombospondin-1 (TSP-1) (FIG. 1L) or IGFBP 1-3 and PF4 (FIG. 1M), ofwhich the latter two were analyzed by protein dot assays using humanangiogenesis arrays (each array contains 56 different angiogenicproteins, R&D Systems, Minneapolis, MN). Briefly, 1.5 ml of human AMextract (25 µg/ml HA) and purified nHC-HA/PTX3(2^(nd)) (25 µg/ml HA)were incubated separately with the detection antibodies pre-coated onthe membrane overnight at 4° C., followed by incubation with thesecondary antibody. Signals were detected with chemiluminescent lightexposed to x-ray film. The array data on developed x-ray film werequantitated by scanning the film on a transmission-mode scanner, and thearray image file was analyzed by ImageJ1.46 software (NationalInstitutes of Health, Bethesda, MD).

Biochemical characterization showed that nHC-HA/PTX3 is composed of highmolecular weight HA (HMW HA) (FIG. 1C) covalently linked to heavy chain1 (HC1) of IαI and PTX3. Both HC1 and PTX3 in nHC-HA/PTX3 are releasedonly after treatment of hyaluronidase (HAase) or NaOH (FIGS. 1D-G),demonstrating that HC1is linked to HA by ester bonds as reported.

In contrast, nHC-HA/PTX3 does not contain HC2 (FIG. 1H), HC3 (FIG. 1I, aband at ~12 kDa detected only after NaOH treatment is likelynon-specific), bikunin (FIG. 1J), TSG-6 (FIG. 1K) and TSP-1 (FIG. 1L).Insulin-like growth factor binding protein-1-3 (IGFBP1-3) and plateletfactor 4 (PF4) are detected by protein dot assay (similar to ELISA) innHC-HA/PTX3(2^(nd)) (FIG. 1J); it remains unclear whether they are stillin nHC-HA/PTX3(4^(th).)

Example 2. Preparation of Immobilized HA (iHA) by Covalent Linkage

A series of hyaluronan (HA) amounts (0, 0.25, 0.5, 1.0, 2.5, 5, 10, and25 µg/well) from HMW HA (Healon, Advanced Medical Optics, Santa Ana, CA)or nHC-HA/PTX3(2^(nd)) was added to the coupling solution containingSulfo-NHS (0.184 mg/ml) and EDAC (0.123 mg/ml) (Both were from ThermoFisher Scientific, Rockford, IL) and incubated in Covalink™-NH 96 wellplates (Thermo Fisher Scientific Inc.), for 16 h at 4° C. After threewashes of 8 M Guanidine-HCl (GnHCl) followed by washes with PBS, thecoupled HA from HMW HA or nHC-HA/PTX3 was measured quantitatively by HAELISA from Corgenix (Westminster, CO) according to the manufacturer’sprotocol (FIG. 6A). HMW HA and nHC-HA/PTX3 purified from AM aredose-dependently and covalently coupled to surfaces of Covalink-NH 96wells. The resultant iHA or immobilized nHC-HA/PTX3 is resistant towashes by 8 M Guanidine HCl. HA of HMW HA or nHC-HA/PTX3 was maximallycoupled at 2 µg/well HA equivalent input (FIG. 6A).

To determine coupling efficiency, HA from HMW HA or nHC-HA/PTX3 wascoupled to Covalink™-NH per well of the 96 well plates, and unbound andbound HA from HMW HA or nHC-HA/PTX3 were measured by HA ELISA (FIG. 6B).2 µg of HA from HMW HA or nHC-HA/PTX3 was added to the coupling solutioncontaining Sulfo-NHS (0.184 mg/ml) and EDAC (0.123 mg/ml)[1-ethyl-3-(3-dimethylaminopropyl) carbodiimide] in H₂O and incubated inCovalink™-NH 96 well plates (Thermo Fisher Scientific, Rockford, IL),for 16 h at 4° C. Both HA coupled to wells or unbound in washed solution(pooled) were measured with HA ELISA from Corgenix (Westminster, CO)according to the manufacturer’s protocol. The total amount of HA in eachwell in either coupled or unbound is divided by the input HA amount (2µg/well) to calculate coupling efficiency or unbound percentage. Theaverage coupling efficiency was determined to be 70.5 ± 13.4 % for HMWHA and 69.0 ± 5.7 % for nHC-HA/PTX3 (FIG. 6B). So, 2 µg/well input HAresults in approximately 1.4 µg iHA.

Example 3. Activity of Purified Native HC-HA/PTX3(nHC-HA/PTX3) ComplexesAttachment of LPS-Stimulated Macrophages to Immobilized nHC-HA/PTX3

RAW264.7 cells (100 µl of 2.5 × 10⁵cells/ml) [American Type CultureCollection (ATCC), Manassas, VA] in DMEM/10% FBS (Life Technologies,Grand Island, NY) were seeded in 96-well plates containing immobilizedHA (Advanced Medical Optics, Santa Ana, CA, 2 µg/well), nHC-HA/PTX3(2µg/well) or PBS control and stimulated with Lipopolysaccharide (LPS) (1µg/ml) (n=3) [LPS-EB Ultrapure, InvivoGen, San Diego, CA].Immobilization of HA and nHC-HA/PTX3 on the surface of Covalink-NH 96well was performed similarly as described above. In brief, Covalink-NH96-well plates were sterilized in 70% alcohol for 2 h, washed 3 timeswith distilled water, and added with 100 µl of 0.184 mg/ml Sulfo-NHS(Thermo Fisher Scientific, Rockford, IL) and 0.123 mg/ml of EDAC (ThermoFisher Scientific, Rockford, IL) in distilled water containing 20 µg/mlHA or nHC-HA/PTX3 per 96-well plate (PBS control wells contain allreagents except for HA and nHC-HA/PTX3). The plate was incubated at 4°C. overnight or at 25° C. for 2 h before the coupling solution wasremoved, washed 3 times with PBS containing 2 M NaCl and 50 mM MgSO4,and followed by 3 washes with PBS. After incubation for 90 min,unattached cells were removed and attached cells were photographed andcounted by the CyQuant assay (FIG. 2A). A greater than 3-fold increasein the attachment of LPS-stimulated macrophages was observed for thewells containing immobilized nHC-HA/PTX3 compared to the control wells.Wells containing immobilized HA inhibited the attachment ofLPS-stimulated macrophages.

The cell viability of attached LPS-stimulated macrophages was thenexamined. LPS-stimulated RAW264.7 cells (100 µl of 2.5 × 10⁵cells/ml)were incubated in DMEM/10% FBS on immobilized PBS control, HA, ornHC-HA/PTX3 for 24 h as described above (n=3). Following incubation, thecell viability of the attached macrophages was measured by MTT assay. Nosignificant differences (all p values > 0.05) in the cell viabilityamong cells on these immobilized substrates were observed (FIG. 2B).

The ability of blocking antibodies and peptides to inhibit attachment ofLPS-stimulated macrophages to immobilized nHC-HA/PTX3 was then examined.RAW264.7 cells (at concentration of 2.5×10⁵cells/ml) were pre-incubatedin DMEM/10% FBS with the blocking antibodies against CD44 (10 µg/ml),TLR2 (10 µg/ml), TLR4 (10 µg/ml), integrin αv (20 µg/ml), β1 (20 µg/ml),β2 (20 µg/ml), or β3 (20 µg/ml) or RGD peptides (SDGRG, RGDS, GRGDS, allat 1 mg/ml), along with the isotype control antibodies [rat IgG (10µg/ml), mouse IgG (10 µg/ml), or Armenian hamster IgG (20 µg/ml)] or aRGD control peptide (1 mg/ml), on ice for 30 min (n=3). (Antibodies toCD44 and rat IgG were from BD Pharmingen, San Diego, CA; antibodies toTLR2, TLR4, and mouse IgG were from InvivoGen, San Diego, CA; antibodiesto integrin αv, β1, β2, β3, and Armenian hamster IgG were fromBiolegend, San Diego, CA; RGD peptides were from Sigma-Aldrich, StLouis, MO). After adding LPS (1 µg/ml), cells were seeded into platescontaining immobilized HA (2 µg/well), nHC-HA/PTX3(2 µg/well) or PBScontrol and incubated for 90 min (n=3). After incubation, unattachedcells were removed and attached cells were photographed and counted bythe CyQuant assay (FIG. 2C). The results showed that antibodies to CD44and TLR4 significantly inhibited attachment of LPS-stimulatedmacrophages demonstrating that these receptors are involved in theattachment of LPS-stimulated macrophages to immobilized nHC-HA/PTX3.

Polarization of LPS-Stimulated Macrophages

Polarization of LPS-stimulated macrophages toward the M1 or M2 phenotypeby immobilized nHC-HA/PTX3 was examined by determining the expression ofgenes encoding M1 and M2 markers by RNA and protein analysis.

RAW264.7 cells (100 µl of 2.5 × 10⁵cells/ml) in DMEM/10% FBS were seededin 96-well plates containing immobilized HA (2 µg/well), nHC-HA/PTX3 (2µg/well) or PBS control and stimulated with LPS (1 µg/ml) for 4 h (n=3).Following incubation, unattached cells were removed and total RNAs wereextracted from the attached cells. The mRNA expression of M1 markers(Tumor necrosis factor alpha (TNF-α) (Mm00443258_m1) and interleukin 12subunit p40 (IL-12p40) (Mm00434165_m1)) and M2 markers (interleukin-10(IL-10) (Mm00439614_m1), Arginase-1 (Arg-1) (Mm00475988_m1),LIGHT/TNSF14 (Mm00444567_m1, and Sphingosine kinase-1(SPHK1)(Mm0044884_g1)) were measured by quantitative real-time PCR withglceraldehyde-3-phosphate dehydrogenase (GAPDH) (Mm99999915_g1) as theendogenous control. The real-time PCR was performed on 7300 Real-timePCR System (Applied Biosystems, Foster City, CA). The amplificationprogram consisted of 10 min of initial activation at 95° C. followed by40 cycles of 15 sec denaturation at 95° C., and 1 min annealing andextension at 60° C. The relative gene expression data was analyzed bythe comparative CT method (ΔΔCT). All assays were performed intriplicate; the results were normalized by GAPDH as an internal control.All primers were from Applied Biosystems. Significant induction ofexpression of the M2 markers IL-10, Arg-1, LIGHT, and SPHK1 compared tothe control was observed in cells attached to immobilized nHC-HA/PTX3,but not HA (FIG. 3A). In addition, expression of both M1 markers, TNF-αand IL-12p40, was reduced.

The amount of secreted TNF-α protein was measured in culturesupernatants of cells treated with LPS (1 µg/ml) stimulation for 4 h inDMEM/10% FBS on plates containing immobilized HA (2 µg/well),nHC-HA/PTX3(2 µg/well) or PBS control as described above (n=3). Theamount of TNF-α was measured by ELISA according to the manufacturer’sprotocol (R&D Systems, Minneapolis, MN).

A reduced amount of TNF-α was observed in the cell culture supernatantsof cells incubated on plates containing immobilized nHC-HA/PTX3 comparedto PBS control (FIG. 3B). No change in the amount of TNF-α was observedon the immobilized HA plate.

High expression of IRF-5 is characteristic of M1 macrophages. IRF-5directly activates transcription of the genes encoding IL-12p40,IL-12p35 and IL-23p19 and represses the gene encoding IL-10. Expressionof IRF-5 protein and its cytolocalization on immobilized nHC-HA/PTX3 wasexamined. Cells seeded on the immobilized control or nHC-HA/PTX3 werestimulated with LPS (1 µg/ml) for 4 or 24 h in DMEM/10% FBS. Theexpression of IRF-5 protein in cell lysates (LPS stimulation for 24 h)was detected by Western blot (FIG. 3C, left) (primary antibody: abcam,Cambridge, MA; secondary antibody, DAKO, Carpinteria, CA). In a parallelexperiment, cells (LPS stimulation for 4 h) were fixed and immunostainedwith anti-IRF-5 antibody. The cytolocalization of IRF-5 was examined byconfocal immunofluorescence microscopy (LSM 700 confocal microscope,Zeiss, Oberkochen, Germany) (FIG. 3C, right). Immobilized nHC-HA/PTX3reduced expression and prevented nuclear localization of IRF-5. Theseresults are consistent with the suppression of the M1 phenotype byimmobilized nHC-HA/PTX3.

Apoptosis of Activated Neutrophils and Macrophage Phagocytosis ofApoptotic Neutrophils

Neutrophils were isolated from the normal human peripheral blood usingthe dextran density [Lymphocyte Poly(R), Cedarlane USA, Burlington, NC ]centrifugation according to the manufacturer’s instruction. Isolatedneutrophils were seeded at 2 × 10⁶ cells/ml in IMDM (Iscove’s ModifiedDulbecco’s Medium, Life Technologies, Grand Island, NY) on immobilizedHA (2 µg/well), nHC-HA/PTX3(2 µg/well) or PBS control and treated withPBS (resting), N-formyl-methionyl-leucyl-phenylalanine (fMLP) (1 µM)(Sigma- Aldrich, St Louis, MO) or LPS (1 µg/ml) for 24 h (n=3).Apoptosis of neutrophils was determined by Cell Death Detection ELISA(Roche Applied Science, Indianapolis, IN) in cell lysates according tothe manufacturer’s protocol. Immobilized nHC-HA/PTX3, but not HA,promotes apoptosis of fMLP or LPS-activated neutrophils but not restingneutrophils (FIG. 3D).

Phagocytosis of apoptotic neutrophils by resting or LPS-stimulatedmacrophages was then examined. RAW264.7 cells (1 × 10⁵ cells/ml) werecultivated in DMEM/10% FBS on the immobilized HA (2 µg/well),nHC-HA/PTX3 (2 µg/well) or PBS control without or with LPS (1 µg/ml)stimulation for 6 days (n=3). The cell culture medium was then removed,and 100 µl of 2 × 10⁶ cells/ml of apoptotic neutrophils in IMDM(prepared by treatment of isolated resting human neutrophils withroscovitine (20 µM) (Sigma- Aldrich, St Louis, MO) for 8 h) were addedto each well containing resting or LPS-stimulated macrophages. Afterincubation for 30 min at 37° C., each well was washed three times withthe cold PBS, and cell lysates (including macrophages and phagocytosedneutrophils) were collected to determine human myeloperoxidase (MPO)activity by the ELISA assay to measure phagocytosed neutrophils bymacrophages. Cell lysates were collected and subjected to humanmyeloperoxidase (MPO) ELISA assay (n=4) (R&D Systems, Minneapolis, MN)according to the manufacturer’s protocol. MPO was then normalized withtotal protein measured by BCA protein assay (Thermo Fisher Scientific,Rockford, IL) in respective cell lysate and expressed as phagocytosisindex. The phagocytosis index of resting cells without LPS (-LPS)stimulation was defined as 100% in this experiment. ImmobilizednHC-HA/PTX3, but not HA, promoted phagocytosis of apoptotic neutrophilsby either resting or LPS-treated macrophages (FIG. 3E).

These results demonstrate that immobilized nHC-HA/PTX3 (2^(nd)) enhancesthe apoptosis of activated neutrophils and phagocytosis of apoptoticneutrophils by macrophages.

Analysis of Receptors Involved in Polarization of M2 Macrophages byImmobilized nHC-HA/PTX3

In order to determine the involvement of particular receptors in M2macrophage polarization, quantitative mRNA expression of M1 and M2macrophage markers in the presence or absence of receptor blockingantibodies was performed. RAW264.7 cells (2.5 × 10⁵ cells/ml) inDMEM/10% FBS were pre-incubated with PBS (control) or blockingantibodies to CD44 (10 µg/ml), TLR4 (10 µg/ml), or CD44/TLR4 (each at 10µg/ml) for 30 min on ice (n=3). Cells were then stimulated with LPS (1µg/ml) and incubated at 37° C. for 4 h on immobilized HA (2 µg/well),nHC-HA/PTX3 (2 µg/well) or PBS control. Total RNAs were extracted fromthe total cells. The relative mRNA expression of M1 marker (IL-12p40)and M2 markers (IL-10, LIGHT, and SPHK1) were determined by quantitativePCR with GAPDH as the endogenous control as described above (FIG. 4A).Expression of IL-12p40 was abolished while that of IL-10, LIGHT andSPHK1 was promoted by immobilized nHC-HA/PTX3, but not HA. Thisexpression pattern was inhibited more by the CD44 blocking antibody thanthe TLR4 blocking antibody. In contrast, expression of IL-12p40 andIL-10 transcript by immobilized HA was affected more by the blockingantibody against TLR4 than that against CD44.

Protein expression of IL-12 and IL-10 also was determined. Cell culturesupernatants were collected from cells cultivated on immobilized HA (2µg/well), nHC-HA/PTX3 (2 µg/well) or PBS control treated as describedabove except for 24 h (instead of 4 h) (n=3). The amount of IL-12 orIL-10 protein in the cell culture supernatants was determined byELISAs(Biolegend, San Diego, CA) according to the manufacturer’sprotocol (FIG. 4B). Expression of IL-12 protein is abolished while thatof IL-10 protein is markedly promoted by immobilized nHC-HA/PTX3. Thisexpression pattern is inhibited by the blocking antibody against CD44.In contrast, expression of IL-12 protein is promoted while that of IL-10is suppressed by immobilized HA, and the expression pattern was affectedmore by the blocking antibody against TLR4.

Comparison of nHC-HA/PTX3(2nd) and nHC-HA/PTX3(4th) Complexes

nHC-HA/PTX3 (2nd) and nHC-HA/PTX3 (4th) complexes were compared byexamining the ability of each complex to induce cell aggregation ofmacrophages (indicative of poor cell attachment) and/or promote M2macrophage polarization. RAW264.7 cells (2.5 × 10⁵cells/ml) werecultivated in DMEM/10% FBS on immobilized HA (2 µg/well), nHC-HA/PTX3 (2µg/well) or PBS control and stimulated with 200 units/ml IFN-γ/1 µg/mlLPS (Both were from InvivoGen, San Diego, CA) for 4 h or 24 h (n=3).After 4 hours, cell aggregation was examined by light microscopy andphotographed (FIG. 5A). Immobilized nHC-HA/PTX3 (4th), but notnHC-HA/PTX3 (2nd), promotes cell aggregation of macrophages, indicatingthat nHC-HA/PTX3 (4th) does not promote cell attachment to the platewhile nHC-HA/PTX3(2nd) does.

After 24 h, samples were obtained from cell culture supernatants andIL-12p40 protein and IL-23 protein concentration was measured byrespective ELISAs (Biolegend, San Diego, CA) according to themanufacturer’s protocol (FIGS. 5B and 5C). Both nHC-HA/PTX3(2nd) andnHC-HA/PTX3(4th) inhibit the production of IL-12p40 and IL-23 proteinsin IFN-γ/LPS -stimulated macrophages.

Example 4: In Vitro Binding of TSG-6 and PTX3 to Immobilized HA (iHA) inthe Absence of IαI Binding of TSG-6 to iHA

Immobilized HA (2 µg/well input) was prepared as described above. Aseries of human TSG-6 (overexpression in mouse myeloma cell line NS0with Trp18 to Leu277 of human TSG-6, with a C terminal 10 His tag,Accession # P98066; R&D Systems, Minneapolis, MN, Cat. No. 2104-TS)concentrations (0, 0.24, 1.2, 6, 12, and 24 µg/ml, 100 µl volume perwell) were incubated with iHA for 2 h at 37° C. in the reaction buffer(5 mM MgCl₂ in PBS, pH 7.5). Unbound TSG-6 was removed by washes of 8 MGuanidine-HCl and PBS. The bound TSG-6 was measured by modified TSG-6ELISA (R&D Systems, Minneapolis, MN). Because TSG-6 was already bound toiHA coupled in wells, the steps of incubating samples with pre-coatedTSG-6 antibody were omitted. The subsequent steps were according to themanufacturer’s protocol (FIG. 7A). TSG-6 dose-dependently bound iHA andreached its maximal binding capacity at about 6 µg/ml (or 0.6 µg in 0.1ml of the reaction solution) when iHA was about 1.4 µg (2 µg HA per wellbased on the coupling efficiency of ~70%). The molar ratio of TSG-6 toHA was about 37:1 based on TSG-6 being 35 kDa and HA being ~3,000 kDa.

The ability of TSG-6/iHA complex to resist dissociation was thenexamined. iHA (2 µg/well input) was prepared as described above. TSG-6(6 µg/ml in 100 µl) was incubated with iHA for 2 h at 37° C. UnboundTSG-6 was removed by washes with PBS (as a control) or with differentdissociating or reducing agents: 6M Guanidine HCl/PBS, 8M GuanidineHCl/PBS, 2% SDS/PBS, 100 mM DTT/PBS, and 25 mM NaOH/H₂O. The bound TSG-6was measured by modified TSG-6 ELISA as described above (FIG. 7B). Theformed TSG-6/iHA complex was stable and was resistant to the treatmentwith the various dissociating or/and reducing agents. No statisticalsignificance was noted among all groups.

Binding of PTX3 to iHA

Immobilized HA (2 µg/well input) was prepared as described above. Aseries of PTX3 (overexpression in mouse myeloma cell line NS0 with Glu18to Ser277 of human PTX3, with a C terminal 6 His tag, Accession #P26022; R&D Systems, Minneapolis, MN) concentrations (0, 0.04, 0.2, 1,5, and 25 µg/ml, 100 µl volume per well) were incubated with iHA for 2 hat 37° C. in the reaction buffer (5 mM MgCl₂ in PBS, pH 7.5). UnboundPTX3 was removed by washes with 8M GnHCl and PBS. The bound PTX3 wasmeasured by modified PTX3 ELISA (R&D Systems, Minneapolis, MN). BecausePTX3 was already bound to iHA coupled in wells, the steps of incubatingsamples with pre-coated PTX3 antibody were omitted. The subsequent stepswere according to the manufacturer’s protocol (FIG. 8A). PTX3dose-dependently bound iHA and reached the maximal binding capacity atabout 5 µg/ml (or 0.5 µg in 0.1 ml of the reaction solution) when iHAwas about 1.4 µg (2 µg HA per well based on the coupling efficiency of70%) The molar ratio of PTX3 to HA was about 24:1 based on PTX3 being 45kDa and HA being ~3,000 kDa.

The ability of PTX3/iHA complex to resist dissociation was thenexamined. iHA (2 µg/well input) was prepared as described above. PTX3 (5µg/ml in 100 µl) was incubated with iHA for 2 h at 37° C. Unbound PTX3was removed by washes with PBS (as a control) or with differentdissociating or reducing agents: 6 M Guanidine HCl/PBS, 8 M GuanidineHCl/PBS, 2% SDS/PBS, 100 mM DTT/PBS, and 25 mM NaOH/H₂O. The bound PTX3was measured by modified PTX3 ELISA as described above (FIG. 8B). Theformed PTX3/iHA complex was stable and was resistant to the treatmentwith the various dissociating or/and reducing agents. No statisticalsignificance was noted among all groups.

Simultaneous Binding of TSG-6 and PTX3 to iHA

iHA (2 µg/well input) was prepared as described above. 6 µg/ml of TSG-6and 5 µg/ml of PTX3 (concentrations for maximal binding as describedabove) were incubated with iHA either alone or combined in the reactionbuffer (5 mM MgCl₂ in PBS, pH 7.5). The bound TSG-6 or PTX3 was measuredby respective modified ELISA as described above. There was nocompetition or synergy for binding to iHA by TSG-6 or PTX3 when bothproteins were incubated with iHA simultaneously as compared to that whenTSG-6 or PTX3 was added alone (p > 0.05). These data indicated that thebinding sites on iHA for TSG-6 and PTX3 are different and might notoverlap (FIG. 9 ).

Sequential Binding of TSG-6 and PTX3 to iHA

Sequential addition of TSG-6 and PTX3 to iHA was examined to determinewhether pre-bound TSG-6 or PTX3 would inhibit binding of the otherprotein to iHA. 6 µg/ml of TSG-6 or 5 µg/ml of PTX3 were pre-bound toiHA, prepared as described above. After washes with 8M GnHCl and PBS,serial concentrations of PTX3 (0, 5, or 5 µg/ml) or TSG-6 (0, 1.2, or 6µg/ml) were subsequently incubated with pre-bound TSG-6/iHA or pre-boundPTX3/iHA in the reaction buffer (5 mM MgCl₂ in PBS, pH 7.5)),respectively. The subsequently bound TSG-6 and PTX3 were measured byrespective modified ELISAs.

Pre-bound TSG-6 (6 µg/ml) partially prevented subsequent PTX3 frombinding to iHA (FIG. 10A) (p = 0.05 and 0.01 when subsequent PTX3 wasadded at1 µg/ml and 5 µg/ml, respectively) (FIG. 5A). Pre-bound PTX3 (5µg/ml) did not interfere with the subsequent TSG-6 binding to iHA (p =0.56 and 0.74 when subsequent TSG-6 was added at 1.2 µg/ml and 6 µg/ml,respectively) (FIG. 10B). These data indicate that iHA is structurallychanged after TSG-6 binding so that it interferes with subsequent PTX3binding.

Example 5: Attachment of LPS-stimulated Macrophages to ImmobilizedTSG-6/iHA and PTX3/iHA Complexes

Covalink-NH 96 wells were covalently coupled with PBS (control), HA(iHA), or native HC-HA/PTX3 (nHC-HA/PTX3) as described above. TSG-6 (6µg/ml) or PTX3 (5 µg/ml) was then added and bound to iHA. RAW264.7macrophages (100 µl of 1 × 10⁵ cells/ml) in DMEM/10% FBS were seededinto each coupled well and treated with 1 µg/ml LPS. After incubationfor 24 h, cell morphology was photographed.

Macrophages attached poorly (i.e., resulting in more aggregated cells)to iHA when compared to the plastic control. Such attachment was furtherhampered by TSG-6/iHA, resulting in high number of and largeraggregations of cells. In contrast, PTX3/iHA promoted cell attachment,resulting in a similar pattern to that shown on immobilized nHC-HA/PTX3(FIG. 11 ).

Example 6: Regulation of M1 and M2 Markers by TSG-6/iHA and PTX3/iHAComplexes

RAW264.7 cells were cultivated as in Example 5 and stimulated with 1µg/ml LPS for 4 h on PBS (control), immobilized HA (iHA), TSG-6/iHA,PTX3/iHA, or nHC-HA/PTX3. Total RNAs were isolated from cells and mRNAexpression of the M2 marker IL-10 and the M1 marker IL-12p40 weremeasured by quantitative PCR (FIGS. 12A and 12D) as described above.Alternatively, the cells were stimulated with 1 µg/ml LPS (FIGS. 12B and12E) or IFN-γ/LPS (FIG. 12C) for 24 h, and protein expression of IL-10,IL-12p70, and IL-23 were measured in cell culture media using respectiveELISAs.

The expression of IL-12p40 mRNA (one of two subunits of IL-12p70) wasnot significantly changed on iHA (none) compared to that of the control(Ctrl) (p > 0.05) (FIG. 12A). In contrast, IL-12p40 mRNA wassignificantly upregulated on TSG-6/iHA (p < 0.01), but significantlydownregulated on PTX3/iHA and nHC-HA/PTX3 (p < 0.05) (FIG. 12A). Theexpression of IL-12p70 protein, however, was only detectable without anysignificant difference (p > 0.05) on the control or iHA alone, but isundetectable on TSG-6/iHA, PTX3/iHA, and nHC-HA/PTX3 (FIG. 12B).IL-12p40 also serves as a subunit of IL-23. It was observed thatexpression of IL-23 protein was significantly upregulated on TSG-6/iHAand PTX3/iHA (p < 0.01), but is undetectable on nHC-HA/PTX3 (p < 0.05)(FIG. 12C). These data indicate that both TSG-6/iHA and PTX3/iHA areeffective in suppressing IL-12 but not IL-23.

The expression of IL-10 mRNA by RAW264.7 cells was not significantlychanged on iHA alone compared to the control (p >0.05), but wassignificantly upregulated on TSG-6/iHA, PTX3/iHA, and nHC-HA/PTX3 (p <0.05) (FIG. 12D). The expression of IL-10 protein, however, is onlysignificantly upregulated on PTX3/iHA similar to the positive control ofimmobilized nHC-HA/PTX3 (p < 0.05) (FIG. 12E). These data suggest thatfollowing different patterns of cell attachment (Example 5), resultantcells exhibit different functions, and that PTX3/iHA is more effectivethan TSG-6/iHA in upregulating IL-10

Example 7: In Vitro Transfer of HC1 and HC2 From IαI to Immobilized HA

Covalink-NH 96 wells were covalently coupled with PBS (control), HA(iHA), or native HC-HA/PTX3 (nHC-HA/PTX3) as described above. SerialTSG-6 concentrations (0, 0.24, 1.2, 6, 12 µg/ml in 100 µl) wereindividually incubated with iHA in the reaction buffer (5 mM MgCl₂ inPBS, pH 7.5)). Human IαI (5 µg/ml) (prepared from human plasma accordingto Blom et al. (1999) J. Biol. Chem. 274, 298-304) was added eithersimultaneously with TSG-6 or sequentially (2 h later). Bound HC1, HC2(antibodies to HC1 and HC2 were from abcam, Cambridge, MA), or IαI(DAKO, Carpinteria, CA) was measured by respective modified ELISAsimilar to TSG-6 and PTX3 ELISAs described above.

The data show that the amount of HC1 (FIG. 13A) or IαI (FIG. 13B) boundto iHA is lower at higher TSG-6 concentrations (6 and 12 µg/ml) whenTSG-6 was pre-bound to iHA with subsequent addition of IαI than whenTSG-6 and IαI were added simultaneously. HC2 was not detected in thesamples (data not shown). The wells were incubated with hyaluronidase todigest the bound HA and to release bound proteins from HA and thesesamples were analyzed by Western blot with anti-TSG-6 antibody (R&DSystems, Minneapolis, MN); the amount of TSG-6 bound to iHA was lowerwhen IαI was simultaneously added with TSG-6 than when IαI wassubsequently added (i.e. after TSG-6 was bound to iHA, FIG. 13C). When 5µg/ml of PTX3 and 5 µg/ml of IαI were incubated with iHA simultaneously,PTX3 but not IαI was bound to iHA (FIG. 13D).

These data indicate that TSG-6 free in solution is more efficient thanTSG-6 bound to iHA in transferring HC1 from IαI onto iHA (FIGS. 13A and13B). More TSG-6 is bound to iHA when TSG-6 is pre-bound to iHA alonethan when TSG-6 and IαI are incubated with iHA simultaneously (FIG.13C), indicating that IαI prevents TSG-6 from binding to iHA if addedsimultaneously with TSG-6, and that TSG-6 might have a higher affinityin binding to IαI than to iHA. In addition, PTX3 free in solution orbound to iHA does not transfer HCs from IαI to iHA (FIG. 13D).

Example 8: Effect of PTX3 on the Formation of HC1•TSG-6 and HC2•TSG-6Complexes

IαI (40 µg/ml) and TSG-6 (6 µg/ml) were incubated in the reaction buffer(5 mM MgCl₂ in PBS, pH 7.5) for 2 h at 37° C. without or with PTX3 (20µg/ml or 120 µg/ml). The reaction samples were analyzed by Western blotwith antibodies against HC1 (FIG. 14A), HC2 (FIG. 14B), TSG-6 (FIG.14C), bikunin (abcam, Cambridge, MA) (FIG. 14D), and PTX3 (data notshown). In solution without HMW HA, TSG-6 forms HC1•TSG-6 and HC2•TSG-6complexes, and generates HMW IαI (FIGS. 14A and 14B). The formation ofHMW IαI is illustrated in FIG. 14E. This data indicates that both HC1and HC2 are transferred by TSG-6 in solution to HMW HA.

Simultaneous addition of PTX3 dose-dependently inhibits formation ofHC2•TSG-6 but not HC1•TSG-6 (FIGS. 14A and 14B). In contrast, HMW IαIcontaining HC2 is increased while HMW IαI containing HC1 is decreased(FIGS. 14A and 14B). TSG-6 forms dimers when added in solution withoutHMW HA with or without PTX3 (FIG. 14C). These findings indicate that 1)both HC1 and HC2 in IαI form either a complex with TSG-6 or a HMW IαIvia TSG-6′s action; 2) PTX3 interferes with the above processdifferently regarding the transfer of HC1 and HC2 by TSG-6. There is notruncated form of HC1 or HC2. The inhibition of HC2•TSG-6 by PTX3 isillustrated in FIG. 14F.

A glycosylated form and a glycosylated and chondroitinsulfate-conjugated form of bikunin about 40 kDa and 45 kDa,respectively, were released by TSG-6 and PTX3, respectively (FIG. 14D).This data is consistent with the published data showing that both TSG-6and PTX3 interact with IαI, and also suggests that TSG-6 and PTX3interact differently with IαI resulting in different outcome of bikunin.

In a separate experiment, HMW HA (250 µg/ml), IαI (40 µg/ml), and TSG-6(6 µg/ml) were incubated in solution for 24 h at 37° C. without or withPTX3 (1, 2.5, and 5 µg/ml) in the reaction buffer (5 mM MgCl₂ in PBS, pH7.5). The reaction samples were analyzed by Western blot with antibodiesagainst IαI (FIG. 14G). In solution with HMW HA but without PTX3, HCsfrom IαI were completely transferred to HMW HA by TSG-6. In the presenceof PTX3, TSG-6-mediated HCs transfer was dose-dependently inhibited,resulting in the accumulation of LMW intermediates (~ 130 kDa and likelyconsisting of HC1-TSG-6) or unprocessed pre-IαI (~130 kDa, unprocessedIαI (220 kDa), and HMW IαI (retained in loading wells) (FIG. 14G). Thesedata are consistent with the finding that PTX3 specifically prevents theformation of HC2-TSG-6, resulting in the inhibition of HC2 transfer andpossible HC1 transfer.

Example 9: Formation of Reconstituted HC-HA/PTX3 (rcHC-HA/PTX3)Complexes in Vitro from Immobilized HA With Simultaneous Addition ofTSG-6, PTX3, and IαI

Immobilized HA (~ 14 µg/ml or 1.4 µg in 100 µl in each well) wasprepared as described in Example 3. IαI (5 µg/ml) and TSG-6 (12 µg/ml)were incubated simultaneously on iHA with or without PTX3 (1, 5, or 20µg/ml) for 2 h at 37° C. the reaction buffer (5 mM MgCl₂ in PBS, pH7.5). After washes with 8 M GnHCl and PBS, bound HC1, TSG-6, and PTX3were measured by respective modified ELISAs (FIGS. 15A, 15D, and 15F,respectively). The wells were washed again with 8 M GnHCl and PBS, andiHA with bound components were digested with 1 unit/ml of hyaluronidasefor 2 h at 60° C. in 10 mM acetate buffer with 75 mM NaCl, pH 6.0. Thesamples were analyzed by Western blot with antibodies against HC1 (FIG.15B), HC2 (FIG. 15C), TSG-6 (FIG. 15E), and PTX3 (FIG. 15G).

Simultaneous addition of TSG-6, PTX3 and IaI to iHA resulted inrcHC-HA/PTX3 complexes containing HMW HC1 but not HC2, and truncated HC1and HC2 (FIGS. 15A-C). PTX3 dose-dependently reduced the amount of HMWHC1 in the complex. The data show that PTX3 dose-dependently interferedwith the transfer of HC1and HC2 to iHA by TSG-6 (FIGS. 15A-C), resultingless HC1/truncated HC1 and truncated HC2 (FIGS. 15B and 15C). TSG-6monomer was decreased while HMW TSG-6 (either multimeric or complexedwith PTX3 and/or HCs) did not change (FIGS. 15D and E).

The data also indicate that PTX3 does not interfere with TSG-6 bound toiHA in the presence or absence of IαI. The published data suggest thatTSG-6 forms dimers with iHA when a smaller MW HA is tested (Baranova etal. (2011) J Biol Chem. 286(29):25675-86). The data present hereinindicate that TSG-6 is complexed in HMW HC-HA/PTX3 complex in thepresence of IαI. Because the free TSG-6 is decreased by PTX3 in adose-dependent fashion, it further indicates that PTX3 promotes thebinding of TSG-6 into HC-HA/PTX3 complex in the presence of IαI. Underthis situation, the majority of PTX3 exists as multimeric forms inHC-HA/PTX3 complex, similar to what has been observed in nHC-HA/PTX3,with a declining amount of monomer, dimers or trimers (FIGS. 15F and G).

Example 10: Effect of Sequential Addition of PTX3 to ReconstitutedHC-HA/PTX3 (rcHC-HA/PTX3) Complexes Formed in vitro With TSG-6 and IαIon Immobilized HA

Immobilized HA (~ 14 µg/ml) was prepared as described in Example 3. IαI(5 µg/ml) and TSG-6 (12 µg/ml) were incubated on iHA in the reactionbuffer (5 mM MgCl₂ in PBS, pH 7.5) for 2 h at 37° C. After removingunbound IαI and TSG-6, reaction buffer with or without PTX3 (1, 5, or 20µg/ml) was incubated with the pre-bound HCs and TSG-6 for 2 h at 37° C.After washes with 8 M GnHCl and PBS, bound HC1, TSG-6, and PTX3 weremeasured by respectively ELISAs (FIGS. 16A, 16D, and 16F, respectively).The wells were then washed again with 8 M GnHCl. PBS control and iHAwith bound components were then digested with 1 unit/ml of hyaluronidasefor 2 h at 60° C. The samples were analyzed by Western blot withantibodies against HC1 (FIG. 16B), HC2 (FIG. 16C), TSG-6 (FIG. 16E),PTX3 (FIG. 16G)

When PTX3 is added subsequently after TSG-6 and HCs have been pre-boundto iHA, PTX3 dose-dependently reduces HC1 transfer to the HMW complex(both intact HC1 and truncated HC1 are reduced) but increases the amountof truncated HC2 in the complex. Consistent with the data shown inExample 7, bound TSG-6 is less efficient than free TSG-6 in transferringHCs to iHA.

Similar to the data shown in Example 8, PTX3 also dose-dependentlyreduced the HMW TSG-6 and monomeric TSG-6 (FIGS. 16D and 16E),indicating that subsequent addition of PTX3 continuously depletespre-bound TSG-6. PTX3, however, is no longer able to be incorporated inTSG-6/HC-HA complex (FIGS. 16F and 16G). Because pre-bound TSG-6 in iHAalso partially prevents PTX3 from binding to iHA (see Example 4), thisfinding indicates that formation of a rcHC-HA/PTX3 complex by TSG-6 andIαI is structurally different from TSG-6/iHA in the extent that PTX3binding to iHA is completely excluded.

Example 11: Formation of Reconstituted HC-HA/PTX3 (rcHC-HA/PTX3)Complexes in Vitro With Pre-Bound PTX3 on Immobilized HA and SequentialAddition of TSG-6 and IαI

Immobilized HA (~ 14 µg/ml) was prepared as described in Example 3. PTX3(5 µg/ml) and iHA were incubated in the reaction buffer for 2 h at 37°C. in the reaction buffer (5 mM MgCl₂ in PBS, pH 7.5). After removingunbound PTX3, the reaction buffer containing TSG-6 (6 µg/ml) and IαI (5,25, and 125 µg/ml) were incubated for 2 h at 37° C. After washes with 8M GnHCl and PBS, bound HC1, TSG-6, and PTX3 were measured byrespectively ELISAs (FIGS. 17A, 17C, and 17E, respectively). The wellswere then washed again with 8 M GnHCl. PBS or iHA with bound componentswere digested with 1 unit/ml of hyaluronidase for 2 h at 60° C. in 10 mMacetate buffer with 75 mM NaCl, pH 6.0. The samples were analyzed byWestern blot with antibodies against PTX3 (FIG. 17B), TSG-6 (FIG. 17D),HC1 (FIG. 17F), and HC2 (FIG. 17G).

In the presence of IαI and TSG-6, pre-bound PTX3 dose-dependentlyincreased ELISA immunoreactivity for PTX3 and the amount of multimericPTX3 but decreased that of monomeric PTX3 in HC-HA/PTX3 complex (FIGS.17A and 17B). This data indicates that multimeric PTX3 promotesimmunoreactivity by this antibody.

Pre-bound PTX3 dose-dependently excluded monomeric TSG-6 whiledecreasing TSG-6 in the rcHC-HA/PTX3 complex (FIGS. 17C and 17D). Thesignificant reduction of bound TSG-6 (both monomer and HMW forms) isdetected when a molar ratio of IaI to TSG-6 is 3:1, where boundmultimeric PTX3 is maximized as well.

There was no significant change in bound HC1based on HC1 ELISA data(FIG. 17E). The transfer of HC2 was dose-dependently increased byincreasing IαI concentrations.

Example 12: Comparison of Macrophage Cell Attachment Activity BetweenReconstituted HC-HA/PTX3 (rcHC-HA/PTX3) Complexes Formed in vitro WithPre-Bound TSG-6 Versus Pre-Bound PTX3 on Immobilized HA

Covalink-NH 96 wells were covalently coupled with PBS (control), HA(iHA), or nHC-HA/PTX3 as described in Example 3. IαI (5 µg/ml), TSG-6 (6µg/ml) or PTX3 (5 µg/ml) were simultaneously or sequentially bound toiHA as follows: (1) (IαI/TSG-6/PTX3)/iHA: IαI, TSG-6, and PTX3 weresimultaneously incubated with iHA for 2 h at 37° C. in the reactionbuffer; (2) (IαI/TSG-6)/PTX3/iHA: IαI and TSG-6 were first incubatedwith iHA for 2 h at 37° C. in the reaction buffer. After removed theunbound IαI/TSG-6, washed with 8 M GnHCl and PBS, PTX3 was added andincubated for 2 h at 37° C. in the reaction buffer; (3)(PTX3)/IαI/TSG-6/iHA: PTX3 was first incubated with iHA for 2 h at 37°C. in the reaction buffer. After removed the unbound PTX3, washed with 8M GnHCl and PBS, IαI/TSG-6 was added and incubated for 2 h at 37° C. inthe reaction buffer. Following formation of the complexes, 100 µl ofRAW264.7 cells (1 × 10⁵cells/ml) were seeded into each coupled well andtreated with 1 µg/ml LPS. After incubation for 24 h, cell morphology wasphotographed.

Macrophages attached poorly to iHA as the control. In the presence ofIαI, simultaneous or pre-bound TSG-6 to iHA ((IαI/TSG-6/PTX3)/iHA or(IαI/TSG-6)/PTX3/iHA) inhibits cell attachment and promotes cellaggregation (FIG. 18 ) similar to the condition without IαI (see Example5). In contrast, pre-bound PTX3 to iHA [(PTX3)/IαI/TSG-6/iHA] promotescell attachment similar to pre-bound PTX3 without IαI as shown inExample 5. The latter resembles the positive control of nHC-HA/PTX3(FIG. 18 ).

Example 13: Comparison of Regulation of M1 and M2 Marker ExpressionBetween Reconstituted HC-HA/PTX3 (rcHC-HA/PTX3) Complexes Formed invitro With Pre-Bound TSG-6 Versus Pre-Bound PTX3 on Immobilized HAExpression of IL-10 and IL-12p40 in Macrophages Cultivated onrcHC-HA/PTX3 Complexes

RAW264.7 cells were cultivated in DMEM/10% FBS on immobilized substratesand stimulated with 1 µg/ml LPS for 4 h as described in Example 12.Total RNAs were isolated and expression of IL-10 and IL-12p40 mRNAs wasmeasured by quantitative PCR as described above (FIGS. 19A and 19C).Alternatively, cells were stimulated with 1 µg/ml LPS for 24 h and IL-10and IL-12p70 proteins in the cell culture supernatants were measured byrespective ELISAs (FIGS. 19B and 19D).

Compared to the PBS control, expression of IL-10 mRNA was notsignificantly changed by iHA (p = 0.56), but was significantlyupregulated on complexes formed by simultaneous addition of TSG-6, IαI,and PTX3 on iHA (IαI/TSG-6/PTX3(a) in FIG. 19 ) (p = 0.0008). Similarly,expression of IL-10 mRNA was significantly upregulated on complexesformed by pre-bound TSG-6 to iHA with subsequent addition of IαI andPTX3 (IαI/TSG-6/PTX3(b) in FIG. 19 ) (p = 0.04) and the positive controlnHC-HA/PTX3 (p = 0.008). Expression of IL-10 mRNA was significantlyhigher on nHC-HA/PTX3 than on IαI/TSG-6/PTX3(a) (p = 0.04), but notsignificantly higher than on IαI/TSG-6/PTX3(b) (p = 0.55). In contrast,expression of IL-10 mRNA was not significantly upregulated by oncomplexes formed by pre-bound PTX3 to iHA (IαI/TSG-6/PTX3(c) in FIG. 19) (p = 0.74) (FIG. 19A). Expression of IL-10 protein, as measured byELISA, was only significantly upregulated by nHC-HA/PTX3 (p = 0.03)(FIG. 19B).

Compared to the control, expression of IL-12p40 (IL-12p40 is one of twosubunits of IL-12p70 and the other subunit is IL-12p35) mRNA was notsignificantly changed by iHA (p = 0.1). In contrast, expression ofIL-12p40 mRNA was significantly upregulated on complexes formed bysimultaneous addition of TSG-6, IαI, and PTX3 on iHA (IαI/TSG-6/PTX3(a)in FIG. 19 ) (p = 0.05) and on complexes formed by pre-bound TSG-6 toiHA (IαI/TSG-6/PTX3(b) in FIG. 19 ) (p = 0.04). In contrast, expressionof IL-12p40 mRNA was completely abolished on complexes formed bypre-bound PTX3 (IαI/TSG-6/PTX3 (c) in FIG. 19 ) and significantlydownregulated by nHC-HA/PTX3 (p = 0.01). There was a statisticalsignificance difference between the latter two conditions (p = 0.04)(FIG. 19C). Compared to the control, expression of IL-12p70 protein wasnot significantly changed by iHA (p = 0.32), but significantlydownregulated on complexes formed by pre-bound PTX3 (IαI/TSG-6/PTX3 (c))(p = 0.03) (FIG. 19D). In contrast, expression of IL-12p70 protein wasabolished on complexes formed by simultaneous addition of TSG-6, IαI,and PTX3 on iHA (IαI/TSG-6/PTX3 (a)), on complexes formed by pre-boundPTX3 (IαI/TSG-6/PTX3 (c)), and nHC-HA/PTX3 (p = 0.05, 0.02, and 0.01,respectively).

Expression of IL-23 in Macrophages Cultivated in the Presence of VariousStimuli

In a separate experiment, IL-23 protein in the cell culture supernatantsof resting RAW264.7 cells (none) or with stimulation of IFN-γ (200units/ml), LPS (1 µg/ml), IFN-γ/LPS, LPS (1 µg/ml) with immune complexor IC (LPS/IC) [IC contained 150 µg/ml IgG-opsonized OVA (IgG-OVA) andwas made by mixing a tenfold molar excess of rabbit anti-OVA IgG(Cappel, Durham, NC) to OVA (Worthington Biochemical Corp., Lakewood,NJ) for 30 min at 25° C.], or IL-4 (10 ng/ml) (R&D Systems, Minneapolis,MN) in DMEM/10% FBS for 24 h was measured. IL-23 protein in the cellculture supernatants was measured by IL-23 ELISA (Biolegend, San Diego,CA) according to the manufacturer’s protocol (FIG. 19E). IL-23 proteinwas undetectable in the cell culture supernatant of resting RAW264.7cells and in those of cells under stimulation for 24 h by LPS (1 µg/ml),LPS with immune complex (LPS/IC), or IL-4 (10 ng/ml), but becamedetectable under stimulation for 24 h by IFN-γ (200 units/ml) and IFN- γ/LPS (FIG. 19E).

Expression of IL-23 in Macrophages Cultivated on rcHC-HA/PTX3 Complexes

In a separate experiment, RAW264.7 cells were cultivated on immobilizedsubstrates as described above and stimulated with IFN-γ/LPS for 24 h.IL-23 in the cell culture supernatants was measured by IL-23 ELISA asdescribed above (FIG. 19F).

Compared to the control, IL-23 protein in the cell culture supernatantof RAW264.7 cells with stimulation of 200 units/ml IFN- γ /1 µg/ml LPSfor 24 h was not significantly affected by iHA (p = 0.02), but wassignificantly upregulated on complexes formed by simultaneous additionof TSG-6, IαI, and PTX3 on iHA (IαI/TSG-6/PTX3 (a)) (p = 0.002) and oncomplexes formed by pre-bound TSG-6 to iHA (IαI/TSG-6/PTX3 (b)) (p =0.0005). In contrast, IL-23 protein is completely abolished on complexesformed by pre-bound PTX3 (IαI/TSG-6/PTX3 (c)) (p = 0.05) similar tonHC-HA/PTX3 (p = 0.05) (FIG. 19F).

Example 14: Use of HC-HA/PTX3 for the Treatment of Chronic Graft VersusHost Disease

Allogeneic hematopoietic stem cell transplantation (HSCT) is apotentially curative treatment for hematological malignancies. However,chronic graft-versus-host disease (cGVHD) remains a major complication.GVHD causes several ocular manifestations in 45-60%, among which dry eyeis the most frequent complication, occurring in nearly 50% of allogeneicHSCT recipients. In fact, dry eye is a distinctive sign and symptom forthe diagnosis of cGVHD. Patients with cGVHD manifest either early-stagemild dry eye disease related to cGVHD or the so called ‘distinctivefeature of cGVHD’ according to the NIH consensus conferenceclassification. Two types of dry eye after HSCT have been noted; one hadsevere ocular surface and tear function damage with decreased reflextearing that occurs soon after the onset of dry eye, whereas the otheris mild with normal reflex tearing. Dry eye typically occurs 6 monthsafter the transplantation and the severity has been reported to becorrelated with the presence of cGVHD and meibomian gland disease. Theonset of cGVHD-related severe dry eye is earlier than that of mild dryeye. For example, severe dry eye occurs 6.8±2.5 months after HSCT, whilemild dry eye occurs 13.2±9.1 months after HSCT. A comparative study of50 eyes of 25 post-HSCT patients and 28 eyes of 14 age-matched healthycontrols showed that MG obstruction, decreased corneal sensitivity,enhanced tear evaporation rate, decreased conjunctival GCD, increasedconjunctival squamous metaplasia and inflammatory cells were noted morein cGVHD-related dry eyes than the normal controls and post-HSCT withoutdry eye subjects. Furthermore, the conjunctival inflammatory cells weresignificantly higher in severe dry eyes compared with mild dry eyes(P<0.03). Moreover, most severe dry eye patients had systemic cGVHD,whereas only a few patients in the mild dry group had systemic cGVHD.Those findings indicated the different pathologic processes incGVHD-related severe and mild dry eye disease. Because comprehensiveocular surface alteration was noted in post-HSCT patients, regardless ofwhether they had cGVHD-related dry eye or not, their results suggestthat the extent of inflammatory process seems to have a pivotal role inthe outcome of the cGVHD-related dry eye. The conjunctival brushcytology specimens showed considerably increased inflammatory cellnumbers in both cGVHD-related severe dry eye and mild dry eye patientscompared with normal controls and post-HSCT without dry eye subjects.Moreover, the number of inflammatory cells in severe dry eye specimenswas significantly higher than in mild dry eye specimens. Furthermore,many inflammatory markers expressed in biopsy samples of the conjunctivaand lacrimal gland from cGVHD-related dry eye patients, confirming thatinflammation is involved in the pathogenesis of cGVHD-related dry eye.

One likely cause of generating a number of scarring complication incGVHD is via EMT of conjunctival basal epithelia and lacrimal glandmyoepithelia as a result of cytokines released by chronic inflammationbecause of infiltrating donor lymphocytes. Previously, it has beenrecognized that inflammation and excessive fibrosis are prominenthistologic features of chronic graft-versus-host disease (cGVHD), butthe mechanism underlying these changes remains unknown. cGVHD manifestsfeatures resembling scleroderma, exhibiting prominent fibrosis in skinlesions, pulmonary fibrosis, and chronic immunodeficiency. Clinicalfeatures of ocular cGVHD include onset of dry, gritty, or painful eyes,cicatricial conjunctivitis including subconjunctival fibrovasculartissue formation, and scleral shortening, which is characteristicfeature of conjunctival fibrosis. In addition to sclerotic features inskin lesions, mucosal atrophy in the mouth, strictures or stenosis inthe upper to mid third of the esophagus, joint stiffness or contracturedue to sclerosis, and bronchitis obliterans in lung together indicatethe characteristic features of systemic GVHD-mediated fibrosis. The mainhistologic findings in the affected exocrine gland and mucosal membraneare marked fibrosis of the interstitium and a prominent increase in thenumber of fibroblasts, accompanied by mild lymphocytic infiltration .Clinically, the severity of the dry eye is correlated with the degree offibrotic change, rather than with the amount of lymphocyticinfiltration, indicating that excessive extracellular matrixaccumulation primarily contributes to the exocrine dysfunction. Thefibroblasts at the interstitium also play a role in inflammation, byattaching to lymphocytes and expressing human leukocyte antigen class IIand costimulatory molecules. These findings together indicate thatfibroblasts play an important role in the pathogenesis of cGVHD.Moreover, we have found that the accumulated fibroblasts in the lacrimalgland of cGVHD patients have a chimeric status. Thus, fibroblastsoriginating from circulating donor-derived precursors and recipientderived fibroblasts may participate in the excessive fibrosis inpatients with cGVHD by interacting T cells. It remains unknown whethercontrolling inflammation by suppressing T cell infiltration will lead toless cicatricial complication in cGVHD.

Previously, donor-derived fibroblasts were detected by combiningimmunohistochemistry and Y-chromosome fluorescent in situ hybridization(FISH) methods in human cGVHD tissue samples. Using a murine model ofcGVHD established by Zhang et al. ((2002) J Immunol. 168:3088-3098), theabove finding can be reproduced. In this model, the tear volume beginsto decrease at 3 weeks after transplantation. Early fibrosis aroundlacrimal gland ducts and progressive fibrosis are detected as early as 3weeks after transplantation, and gradually progress for up to 8 weeks ina similar fashion to human samples. We have performed this experimentsover 20 times to create both GVHD and control groups with success,resulting in overall reproducibility is 70-80% based on analysis oflacrimal gland tissue samples and tear volumes.

In a typical transplantation experiment, 7- to 8-wk-old male and femaleB10.D2 (H-2d) and BALB/c (H-2d, Sankyo Laboratory, Ltd) mice are used asdonors and recipients, respectively, using added spleen cells as asource of mature T cells. Briefly, female recipient mice are lethallyirradiated with 700 cGy from a Gammacel 137Cs source (J. L.Shepherd &Associates, San Fernando, CA). Approximately 6 h later they are injectedby tail vein with male donor bone marrow (1 × 10⁶/mouse) and spleen (2×10⁶/mouse) cells suspended in RPMI 1640 (BioWhittaker, Walkersville,MD). A control group (syngeneic BMT) consists of female BALB/c recipientmice that receive the same number of male BALB/c spleen and bone marrowcells. (Zhang et al. (2002) J Immunol. 168:3088-3098). For HC-HA/PTX3treatment, HC-HA/PTX3 complexes are administered via subconjunctivalinjection at predetermined times following bone marrow transplantation,such as 7, 14, 21 and 28 days following bone marrow transplantation.

Effects of treatment are assessed using assays including, but notlimited to measurement of lacrimal gland fibrosis using Mallorystaining, determining the number of activated fibroblasts per fieldusingHSP47, a collagen specific molecular chaperon, as a marker ofactivated fibroblasts, measure lacrimal tear production underpilocarpine stimulation using a cotton thread test, and determining thelevel of fibrogenic cytokines such as HSP47, IL-4, IL-6, and TGF-betausing RT-PCR.

It is expected that treatment with HC-HA/PTX3 complexes will result inthe reduction of lacrimal gland fibrosis in the mouse model. HC-HA/PTX3complexes are then administered in the clinical setting by means ofsubconjunctival injection to human subjects for the treatment of dry eyecaused by cGVHD.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 15: Use of HC-HA/PTX3 for the Treatment of Inflammation in aMouse Model

In this example, anti-inflammatory efficacy is tested in a murine modelof HSV-1 necrotizing corneal stromal keratitis. A total of 240 femaleBALB/c mice (6 - 8 week old) obtained from Charles River Wiga (Sulzfeld,Germany) are anesthetized by intraperitoneal injection of 2 mg ketamineHCl and 400 ng mepivacaine HCl. For each mouse, the central cornea ofone eye is then be scratched in a crisscross pattern with 8 horizontaland 8 vertical scratches using a 27-gauge needle under a surgicalmicroscope. Each injured cornea is applied with a 5 µl suspensioncontaining 1×10⁵ plaque forming units of HSV-1 viruses (KOS strain),which are routinely propagated on Vero cells, stored at -80° C., andquantified by standard plaque assay. On Day 14 after HSV-1 inoculation,mouse corneas that have developed severe ulcerating stromal keratitisare included for the study (about 50% yield) and subdivided into threegroups, each consisting of 40 corneas (n=6 for clinical examination, n=5for histology, n=5 for immunostaining, n=6 for cytokine ELISA, n=5 forTUNEL, and n=10 for flow cytometry, and n=3 for attrition/backup). Thenon-infected fellow eyes are used as the negative control group. Thepositive control group receive tarsorrhaphy using 2 10-O nylon suturesto close eyelids. The experimental HC-HA/PTX3 group receive the sametarsorrhaphy as the positive control and topical application 4 times aday of a composition containing purified HC-HA/PTX3 complex. Theexperimental HA group receives the same tarsorrhaphy but topicalapplication of composition of HA alone four times a day. After 2 days,tarsorrhaphy is removed in all three groups. Using an operationmicroscope (Zeiss, Germany), the severity of stromal inflammation ofeach cornea is evaluated by a score of 0 to 4+, with 1+ having less than25%, 2+ less than 50%, 3+ less than 75%, and 4+ between 75 and 100%corneal opacity with corneal neovascularization, edema, and thinning.After euthanasia by CO₂ chamber followed by cervical dislocation, 5corneas from each group are subjected to frozen sectioningimmunostaining using primary antibodies to CD11b (neutrophils andmacrophages), F4/80 (macrophages), Gr-1 (PMNs), and CD3 (T cells) (seeMethods), and another 5 corneas from each group are submitted tohematoxylin-eosin staining and TUNEL staining. In addition, cornealhomogenates prepared from 6 corneas from each group are subjected toELISA measurement of IL-1α, IL-2, IL-6, IFN-γ and TNFα levels. Cellsreleased by collagenase from 10 corneas of each group are prepared forflow cytometry to quantitate viable cells by MTT assay and apoptoticcells by the Annexin V-PE Apoptosis Detection Kit (BD-Pharmingen,Heidelberg, Germany).

It is expected that 50% mouse HSV-1-infected corneas will develop severecorneal stromal keratitis (inflammation), edema, and ulceration in twoweeks after inoculation to be included for the study. Two days later,the non-infected corneas will remain normal, while infected corneas inthe control group will maintain similar severe inflammation whentarsorrhaphy is removed. Similar to the control group, corneas in theexperimental HA group will exhibit similar severe inflammation. Incontrast, corneas of the experimental HC-HA/PTX3 group will showreduction of inflammation, which will be correlated with andsubstantiated by significant reduction of inflammatory (PMN/macrophages)and immune (T-cells) infiltration based on histology and immunostainingto CD11b, F4/80, Gr-1, and CD3, by significant reduction of inflammatoryand immune cytokines such as IL-1α, IL-2, IL-6, IFN-y, and TNF-α basedon ELISA and by a significant increase of TUNEL-positive cells incorneal tissues and of dead (MTT) and apoptotic cells released bycollagenase from the cornea (flow cytometry using Annexin-V/7-AAD) whencompared to the positive control group and the experimental HA group.Collectively, these data support the notion that the HC-HA/PTX3 complexexerts a clinical anti-inflammatory efficacy in this murine HSV-1 model.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 16: Use of HC-HA/PTX3 for the Inhibition of Scarring in a RabbitModel

In this example, anti-scarring efficacy tested in a rabbit model ofexcimer laser-assisted photorefractive keratectomy (PRK). A total of 30New Zealand white albino rabbits with body weight (BW) of 2.5-3.0 kg,either sex, are used and subdivided into three groups (n=10 each): thenon-PRK control group, the PRK HA group, and the PRK HC-HA/PTX3 group.Before PRK and for all CMTF examinations, rabbits are anesthetized byintramuscular injection of 5 mg/kg BW xylazine and 30 mg/kg BW ketamineand topically by 0.5% tetracaine HCl ophthalmic solution (OrtopicsLaboratories Corp., Fairton, NJ). For the two PRK groups, the cornealepithelium of one eye of each animal is manually be removed by gentlescraping with a blunt spatula in an area just larger than the ablationzone, the denuded stroma is irrigated with normal saline, and excessfluid is removed gently with a cellulose sponge. A standard 6 mmdiameter, 9.0 D PRK myopic correction PRK is performed using aLaddarVision Excimer Laser (Alcon, Ft. Worth, TX) to achieve a predictedtheoretical stromal ablation depth of 118 µm. Immediately following PRKand thereafter, the PRK HC-HA/PTX3 Group is applied with a compositioncontaining the HC-HA/PTX3 complex while the PRK HA group is applied withcomposition containing HA alone, both four times a day thereafter for atotal of 3 weeks. In addition, all PRK-treated eyes is instilled topical0.1% sodium diclofenac (one drop immediately post-PRK) and 0.3%gentamicin sulfate (three times daily for 3 days).

In vivo CMTF is performed on all operated eyes (n=6 from each group)prior to PRK and at one, two, three, and 4 weeks, two months and 4months post-PRK using a modified Tandem Scanning Confocal Microscope(Tandem Scanning Corporation, Reston, VA) with a 24X surface-contactobjective. Following a standard confocal examination of cornealmorphology, video camera setting (gain, kilovolts, and black level) areswitched to manual and kept constant during the study to allow directcomparison of all scans. CMTF is performed as a continuous, z-axis scanthrough the entire cornea. Corneal, epithelial, and stromal thicknessare mapped within the central 3 mm zone by performing 10 consecutiveCMTF-scans in areas covering all regions. Only data obtained from thethinnest stromal region corresponding to the center of the photoablationprofile are used for subsequent calculations. CMTF-profiles based onimage intensity depth are generated from CMTF videorecordings. Corneallight reflectivity is measured by CMTF-profiles and expressed inarbitrary units (U) defined as µm*pixel intensity as an estimate ofcorneal haze.

To identify and measure the presence of stromal fibrotic tissue, threePRK-treated animals from each group (control and treated) are vitallystained with 0.5% 5-(4,6-dichlorotriazinyl) aminofluorescein (DTAF)dissolved in 0.2 M sodium bicarbonate as previously reported. After 2min staining, eyes are thoroughly rinsed to remove excess dye beforeadministration of topical antibiotics. At 4 months post-PRK animals iseuthanized by intraveneous injection of sodium pentobarbital (120 mg/kgBW). Following euthanasia, all corneas are fixed in situ by anteriorchamber perfusion of 2% paraformaldehyde in PBS, pH 7.2, for 3 min,excised, placed in fresh fixative, and stored at 4° C. Tissue is then byembedded in OCT, snap frozen in liquid nitrogen and sectioned using acryomicrotome. Tissue is serially stepped section to identify thecentral and deepest part of the photoablation, and subjected toimmunostaining using antibodies to keratocan, CD3434, FITC-conjugatedphalloidin, ED-A fibronectin, S-100A4 and α-smooth muscle actin (α-SMA)to correlate changes of corneal haze (by CMTF light reflectivity) withphenotypic changes from keratocytes to fibroblasts or myofibroblasts.Additionally, in those eyes that are stained with DTAF, the thickness ofthe fibrotic tissue that is deposited is measured by determining thedistance between the basal epithelial cells and the DTAF stained cornealtissue which represents the original, undamaged corneal stroma.

It is expected that in vivo confocal microscopy will revealcharacteristic epithelial, basal lamina, stromal and endothelialcharacteristics, which will correlate well with well-defined peaks thatchange in intensity and position over time when in vivo CMTF-profilesare analyzed for corneas of the non-PRK control as well as for thosereceiving PRK. According to the published data, the PRK-treated corneaswill exhibit four peaks originating from the superficial epithelium,photoablated stromal surface, layers of spindle-shaped fibroblasts, andendothelium, while those of the non-PRK control will exhibit three peaksoriginating from superficial epithelium, basal lamina, and endotheliumat one week post-PRK. It is expected that there will not be muchdifference between the two experimental groups at one week post-PRK. At2 weeks post-PRK, the experimental PRK HA group will show an increasingintensity of the peak close to the photoablated stromal surface due toongoing cell migration of spindle-shaped fibroblasts. However, it isexpected that the intensity of the repopulating fibroblasts (by theheight of the peak) will be much reduced in the experimental PRKHC-HA/PTX3 group. During the period from 3 weeks to four monthspost-PRK, the peak corresponding to the layer of spindle-shapedfibroblasts will merge with the peak originating from the photoablatedstromal surface because of the completion of repopulation of theacellular anterior stroma in the experimental PRK HA group, and willresult in a dramatic increase of the reflectivity of the peakcorresponding to the photoablated stromal surface. In contrast, therewill not be such a dramatic increase of the reflectivity in theexperimental PRK HC-HA/PTX3 group. Such a difference of lightreflectivity can also be quantitated by calculating the area of theCMTF-peaks originating from specific intra-corneal structures. It isexpected that the experimental PRK HA group will exhibit a substantiallinear increase in reflectivity intensity within the first 2 to 3 weekspost-PRK and a slow linear decline in reflectivity thereafter. Incontrast, it is expected that there will be a significant decrease ofreflectivity in the experimental PRK HC-HA/PTX3 group during bothperiods. Collectively, these CMTF data support that HC-HA/PTX3 complexexerts an inhibitory effect of keratocytes activation, migration andcell recruitment during repopulation of the acellular anterior stroma,explaining why corneal light scattering (haze) is reduced similar toanti-TGF-β antibodies previously reported. As a result, there is lesscellularity and reflectivity of activated, migrating, intra-stromalwound healing keratocytes, and less deposition of new stromalextracellular matrix and a faster establishment of a normal quiescentkeratocytes population in the anterior stroma. This conclusion will becorroborated by a significant reduction of activated keratocytes(F-actin in keratocan expressing cells), fibroblasts (cytoplasmicstaining of S-100A4, membrane expression of ED-A fibronectin), andmyofibroblasts (nuclear expression of S100A4 and cytoplasmic expressionof α-SMA) in the experimental PRK HC-HA/PTX3 group when compared to theexperimental PRK HA group during the period of 2 to 3 weeks post-PRK. Italso is expected that there will be a significant reduction of thedistance between the basal epithelial cells and the DTAF stained cornealtissue in the PRK HC-HA/PTX3 group, indicative of significantly lessfibrotic tissue, when compared to the PRK HA group.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 17: Use of HC-HA/PTX3 for the Treatment of Atherosclerosis

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of atherosclerosis.

Atherosclerosis includes the involvement an inflammatory cellpopulation, in particular macrophages. A macrophage phenotypic switch isobserved during disease progression. In atherosclerosis, circulatingmonocytes are recruited to sites of fatty deposit accumulation withinthe vascular intima and subintima via CCR2 and endothelial adhesionmediated mechanisms. Upon arrival these cells become activated anddifferentiate into macrophages. The fatty deposits then begin to matureinto plaques with continued recruitment of inflammatory cells, smoothmuscle cells, and the production of extracellular matrix. The initialinfiltrating macrophage population in early atherosclerosis isheterogeneous, but possesses a predominantly M2-like phenotype.Concurrent with lesion progression and expansion, a switch to apredominantly M1 phenotype has been observed. This phenotypic switch maybe due to the phagocytosis of excess oxidized low-density lipoproteins(LDL) within the plaque by macrophages and the production of IFN-γ bylocal Th1 cells, resulting in the development of foam cell macrophages.Foam cell macrophages exhibit a highly activated phenotype leading toproduction of pro-inflammatory mediators and MMPs that destabilize theplaques, potentially leading to thromboembolism. Therapies which preventthe M2 to M1 switch or deplete M1 macrophages selectively are ofclinical utility for the stabilization of atherosclerotic plaques.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject having atherosclerosis. The HC-HA/PTX3 complexemployed, for example, to coat implantable medical devices, such as astent, for implantation in at or near the site of inflammation.Treatment of atherosclerosis with an HC-HA/PTX3 complex is expected todecrease inflammation and prevent thromboembolism.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 18: Use of HC-HA/PTX3 for the Treatment of Obesity and InsulinResistance

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of obesity andinsulin resistance.

Adipose tissue macrophages (ATM) comprise a significant proportion ofthe cellular component of adipose tissue in both lean and obese states.In normal humans, ATMs make up as much as ten percent of the cellularconstituents of the tissue. In comparison, in obese subjects that numberrises to as much as 40%. In normal, non-obese subjects, ATMs have apolarized M2 phenotype characterized by increased baseline STAT6 andPPAR-γ expression. These cells play an important and beneficial role innutrient metabolism. The deficiency in PPAR-γ leads to impaired M2macrophage function and susceptibility to diet-induced inflammation andinsulin resistance. In contrast, ATMs accumulate in the adipose tissueduring obesity have a strongly polarized pro-inflammatory M1 phenotype.These cells produce high levels of TNFα, IL-6, and IL-1β, all of whichare also observed in increased levels of adipose tissue from insulinresistant individuals. High levels of pro-inflammatory mediators locallyimpair the function of resident insulin processing cells.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject suffering from obesity or insulin resistance.An HC-HA/PTX3 complex is administered, for example, as a solution of gelfor treatment. It is expected that treatment with an HC-HA/PTX3 complexwill promote a phenotypic switch of adipose tissue macrophages (ATM)from a pro-inflammatory M1 phenotype to an M2 phenotype and restorenormal insulin processing.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 19: Use of HC-HA/PTX3 for the Treatment of Type 1 Diabetes

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of type 1 diabetes.

Diabetes mellitus type 1 (Type 1 diabetes, T1DM, IDDM, or, formerly,juvenile diabetes) is a form of diabetes mellitus that results fromautoimmune destruction of insulin-producing beta cells of the pancreas.The subsequent lack of insulin leads to increased blood and urineglucose. The classical symptoms are polyuria (frequent urination),polydipsia (increased thirst), polyphagia (increased hunger), and weightloss.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject suffering from Type 1 diabetes in the form ofa microcapsules containing autologous or allogeneic insulin-producingcells coated with HC-HA/PTX3 complex. The microcapsules are administeredto a subject for example, by injection. It is expected that treatmentwith the HC-HA/PTX3 coated microcapsules will allow for the productionof insulin that is released in the subject and prevent or reduceinflammatory responses against the cell therapy or microcapsule, therebyalleviating the Type 1 diabetes and the symptoms thereof.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 20: Use of HC-HA/PTX3 for the Treatment of Fibrosis

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of fibrosis orfibrotic disorder.

The progressive fibrotic diseases, such as idiopathic pulmonary fibrosis(IPF), hepatic fibrosis and systemic sclerosis, are tightly regulated bymacrophages. ‘Pro-fibrotic’ macrophages exhibit M1 properties andproduce various mediators, including TGFβ1, PDGF and insulin-like growthfactor 1, that directly activate fibroblasts and myofibroblasts, whichcontrol ECM deposition. Pro-fibrotic macrophages also produce MMPs,TIMPs, and IL-1β. IL-1β stimulates TH17 cells to produce IL-17, animportant inducer of bleomycin-induced pulmonary fibrosis, a fibroticdisorder with characteristics that are similar to those of IPF. Theproduction of IL-10, RELMα and ARG1 by M2-like macrophages suppressfibrosis.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject suffering from fibrosis or a fibroticdisorder. An HC-HA/PTX3 complex is administered, for example, as asolution, gel or as a coating on an implantable medical device. It isexpected that treatment with an HC-HA/PTX3 complex will decrease the M1macrophages and activation of fibroblasts and myofibroblasts andincrease the amount M2 macrophages present at the affected site(s) inthe subject thereby suppressing the fibrosis and symptoms thereof, suchas scarring.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 21: Use of HC-HA/PTX3 for the Treatment of Chronic Inflammation

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of a chronicinflammatory condition, such as rheumatoid arthritis.

Many autoimmune diseases, including rheumatoid arthritis, involveinflammatory responses to autoantibodies that activate Fc receptors totrigger mast-cell and macrophage activation,_and neutrophil invasion.This leads to an intense local inflammatory response and, if notresolved, to tissue_damage over time with cycles of repair anddestruction. In rheumatoid arthritis, CSF1 is produced constitutively bysynovial fibroblasts and recruits tissue-infiltrating monocytes andmacrophages. In addition, locally produced CSF1, together with RANK1,induces the differentiation of monocytes to osteoclasts, which triggerbone loss.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject suffering from a chronic inflammatorycondition, such as rheumatoid arthritis. An HC-HA/PTX3 complex isadministered, for example, as a solution, gel or as a coating on animplantable medical device. It is expected that treatment withHC-HA/PTX3 will suppress M1 proinflammatory macrophages, induceneutrophil apoptosis, and inhibit osteoclast differentiation, therebytreating the inflammatory condition and the symptoms thereof.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 22: Use of HC-HA/PTX3 for the Treatment of Acute InflammatoryResponse

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of an acuteinflammatory response caused by a condition such as myocardialinfarction, stroke or sepsis. An HC-HA/PTX3 complex generated by themethods described herein is administered to a subject having an acuteinflammatory response caused by a condition such as myocardialinfarction, stroke or sepsis. An HC-HA/PTX3 complex is administered, forexample, as a solution by intravenous infusion. It is expected that theHC-HA/PTX3 complex will decrease or prevent damage caused by acuteinflammation by suppression of M1 inflammatory macrophages.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 23: Use of HC-HA/PTX3 for the Treatment of Cancer

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of cancer.

The participation of large numbers of inflammatory cells in tumordevelopment and progression has been observed and is commonly describedas “smoldering inflammation”. These observations have led to awell-established link between inflammatory cells, macrophages inparticular, and cancer. It was initially thought that development ofoncogenes resulted in the hallmark microenvironment of cancer, in whichtransformed cells secrete cytokines and chemokines that promote tissuedevelopment and prevent apoptosis as well as suppress cytotoxic immuneresponses (termed the “intrinsic pathway”). It is now recognized thatanother pathway leading to tumorigenesis exists. This “extrinsicpathway” is initially characterized by a chronic pro-inflammatoryenvironment resulting from a persistent microbial infection, autoimmunedisease, or other etiology of unknown origin . The chronic production oflarge quantities of inflammatory mediators in these cases can lead totumor cell proliferation and survival or to the induction of geneticinstabilities in normal cells, with resultant expression of oncogenesand production of immune suppressive cytokines . Thus, early tumordevelopment is, in many instances, characterized by a polarizedinflammatory, M1-like macrophage environment.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject having a cancer, such as a solid tumor cancer.An HC-HA/PTX3 complex is administered, for example, as a solution, gelor as a coating on an implantable medical device for topical, injective,or implantive application. Because HC-HA/PTX3 can suppress M1 macrophagepolarization, it is expected that treatment with HC-HA/PTX3 will inhibitor prevent cancers or their progress into late stage phenotypes.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 24: Use of HC-HA/PTX3 for the Treatment of Non-Healing SkinWounds or Ulcers

In this example, an HC-HA/PTX3 complex generated by the methodsdescribed herein is administered for the treatment of a non-healingwound or ulcer on the skin.

A non-healing wound or ulcer on the skin that has been present for about3-4 weeks duration, without healing is called non-healing ulcer.Diseases that commonly cause non-healing ulcers are vascular disease,diabetes, skin cancers and some infections.

An HC-HA/PTX3 complex generated by the methods described herein isadministered to a subject having a non-healing wound or ulcer on skin.An HC-HA/PTX3 complex is administered, for example, as a solution, geltopically or subcutaneously for the treatment at the site of the woundor ulcer. It is expected that treatment with HC-HA/PTX3 will promote thehealing of the wound or ulcer by promoting the M2 phenotype of woundhealing and tissue regenerative macrophages.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 25: Use of HC-HA/PTX3 for the Treatment of High Risk CornealTransplants

In this example, HC-HA/PTX3 is administered for the treatment of highrisk corneal transplants. Mafia mice, which EGFP+ macrophages areintrastromally injected with LPS (5 µg per eye) for both eyes. In eacheye, OS (oculus sinister; left eye) is treated with PBS (2 or 4injection sites) while OD (oculus dexter, right eye) is treated one timewith HC-HA/PTX3 (2 or 4 injection sites; 5 µl of 1 mg/ml HA compositioncontaining HC-HA/PTX3 per injection site) immediately after LPSinjection. Images of whole corneas are taken with in vivo intravitalmicroscopy on day 1, day 2, day 3, day 4, day 5, day 6, and day 7.EGFP-positive cells are counted based on the intensity of greenfluorescence to determine the level of EGFP infiltration. In a mousemodel of corneal transplantation, injection of HC-HA/PTX3 intosubconjunctival sites is expected to reduce inflammation (i.e.infiltration of macrophages) and improve the survival rate oftransplanted corneas when compared to PBS vehicle control.

In some examples, the HC-HA/PTX3 complex used in the method of treatmentdescribed herein is an isolated native HC-HA/PTX3 complex (nHC-HA/PTX3).In some examples, the nHC-HA/PTX3 is isolated from umbilical cordtissue. In some examples, the nHC-HA/PTX3 is isolated from amnioticmembrane. In some examples, the HC-HA/PTX3 complex used in the method oftreatment described herein is a reconstituted HC-HA/PTX3 complex(rcHC-HA/PTX3).

Example 26: Distribution of HA, PTX3, TSG-6, HC1, HC2, HC3 and Bikuninin Umbilical Cord (UC)

In this example, the in vivo distribution of HA, PTX3, TSG-6, HC1, HC2,HC3 and bikunin was detected in umbilical cord (UC) by immunostaining.UC tissue frozen sections were subjected to immunostaining for HA, PTX3,TSG-6 and various components of IαI including HCs and bikunin. UCconsisted of a layer of epithelium and a stroma composed of a sub-amnionlayer and a Wharton’s jelly that contained three vessels, i.e., one veinand two arteries (FIG. 20 a , with one artery vessel, phase). Strongpositive HA staining was observed in the UC epithelium, sub-amnion layerand Wharton’s jelly, and weak HA staining in the vessel wall (FIG. 20 a, HA). With HAase digestion, the aforementioned HA staining disappeared(FIG. 20 a , HA (+ HAase)), conforming the specific staining for HA.

Strong positive immunostaining of PTX3 was present in Wharton’s jelly,and weak PTX3 staining in sub-amnion layer and epithelium (FIG. 20 a ,PTX3). HAase digestion did not enhance PTX3 staining in the sub-amnionlayer and the epithelium (not shown), suggesting that the weak PTX3staining was not due to the masking effect by HA. Positive PTX3 stainingwas also observed in the endothelium of vessels (not shown) but not inthe vessel wall of arteries and vein.

Both TSG-6 and bikunin were present in the whole UC with TSG-6 mainlypresent in cells and around cells and more TSG-6 was in epithelium andsubamnion compared with Wharton’s jelly. HC1 had a similar localizationas HA except that the epithelium and the vessel wall had faint HC1staining. Weak to no HC2 and HC3 staining was present in the epitheliumbut not in the stroma of UC. These results showed that UC producedabundant HA, PTX3, TSG-6, HC1 and bikunin, and disproportionally lessHC2 and HC3 when compared to AM. It was determined that UCconstitutively expressed the above proteins, and HC-HA/PTX3 complex.

Furthermore, PTX3 was present in UC with a different distributionpattern from what has been reported in AM. More PTX3 was present in theUC Wharton’s jelly and less in epithelium and sub-amnion. In contrast,more PTX3 was in the epithelium and the stromal compact layer in AM. UChad a similar pattern to AM in the following markers: more HA waspresent in the whole stroma of UC and little in epithelium of UC. Thiswas similar in the distribution pattern of HA in AM. TSG-6 was mainlylocalized in epithelial and subamnion cells of UC, and bikunin was foundin the whole UC.

Example 27: Comparison of the Extracts of PBS and GnE SequentiallyObtained From AM and UC

This example determined whether the insoluble part after PBS extractfrom AM still contained any PTX3, TSG-6 and IαI as well as HC-HA/PTX3complex. Proteins were extracted from the insoluble part of AM by 4 MGnHCl after PBS extraction to see whether there was PTX3, TSG-6 and IαI.In addition, UC with PBS was also sequentially extracted with 4 M GnHClto detect PTX3, TSG-6, HCs and bikunin in these two differentextractions.

According to the method described in He et al. (2009) J. Biol. Chem.284:20136-20146), AM, CH and UC were homogenized with a blender in coldPBS at 1:1 (g/ml) for AM or 1:1.5 (g/ml) for UC, and mixed at 4° C. for1 h. The mixture was centrifuged at 48,000 g at 4° C. for 30 min. Thesupernatants of PBS extract were designated as AME, CHE and UCE,respectively. In addition, Wharton’s jelly mixture from UC was alsoextracted by PBS and such extract was named UJE. The insoluble pellet ofAM, CH, UC and UC jelly mixture after PBS extraction were furtherextracted by 4 M GnHCl buffer (100 mM sodium acetate, pH 5.8, 4M GnHCl,10 mM EDTA, 1% Triton X-100) at 4° C. for 24 h. After centrifugation at48,000 g, at 4° C. for 30 min, the supernatants were collected and namedAMGnE, CHGnE, UCGnE and UJGnE, respectively. The HA and proteinconcentrations in each extraction were detected by HA ELISA and BCAassay, respectively.

GnHCl Further Extracted Abundant HA and Proteins From the InsolublePellet after PBS Extraction

The HA and protein concentrations in sequential PBS and GnHCl extractsare summarized in Table 1 where the HA/protein ratio was also comparedbetween the two extracts. In general, 4 M GnHCl further extractedabundant proteins and HA from the insoluble pellet of AM, CH, UC and UCjelly mix after PBS extraction. GnHCl buffer extracted more proteins butless HA from the insoluble pellet than PBS. However, UCGnE stillcontained similar amount of proteins and HA to AME and CHE. That is, UCcontained more HA than AM and CH in both PBS and GnHCl extracts.

TABLE 1 Quantitation of proteins and HA in 4 M GnHCl extracts (GnE) frominsoluble pellets of AM, CH, UC and UC jelly mix after PBS extract HA (µg/ml) Protein ( µg/ml) HA/protein Ratio (µg/µg) AME I103 75.7 1014.30.074633 AME J021 61.5 5353.0 0.011489 AMGnE I103+J021 47.7 6097.50.007823 CHE I103 78.9 6161.7 0.012805 CHGnE I103 24.9 7021.7 0.003546UCE 1 I103 453.5 8523.8 0.053204 UCGnE 1 I103 88.2 1925.7 0.045802 UCE 25001 421.3 7471.3 0.056389 UCGnE 2 5001 79.6 2670.2 0.029811 UCJE 277.55135.1 0.05404 UCJGnE 57.2 4955.1 0.011544

Monomer, Dimer, and HMW PTX3 Was Present at Higher Amounts in PBSExtraction of UC Cells Compared to AM Cells. But HMW PTX3 Was Present inHigher Amounts in GnHCl Extraction in AM Cells

Analysis of the AME with anti-PTX3 antibody revealed a band of ~45 kDacorresponding to the size of the native PTX3 monomer, and a HMW band atthe bottom of the loading well (FIG. 21A, lane 4). NaOH treatment didnot affect the 45 kDa band, but completely eliminated the HMW band,resulting in a HMW smear of PTX3 (FIG. 21A, lane 5), which is a notablefeature of PTX3 in HC-HA complex with NaOH treatment where no monomerPTX3 but a 90 kD dimer was detected with or without NaOH treatment (FIG.21A, lane 2 and 3). The HMW smear of PTX3 represented the complexesformed between PTX3 and HC-HA. CHE had the same pattern of PTX3 band,but there was less of a smear of PTX3 after NaOH treatment, consistentwith the immunostaining result. Placenta extract had the same resultwith CHE. Notably, PTX3 existed more in a dimer and HMW smear besidesmonomer in UCE, and their intensity further increased after NaOHtreatment. Similar to AME, UCE also generated HMW smearing pattern, moreso than AME. These results showed that UCE contain more PTX3 than AME,while CHE and placenta extracts contained little PTX3.

Compared with AME (FIG. 21A), AMGnE showed a strong HMW PTX3 smear, weakdimer and monomer levels of PTX3, and the intensity of HMW PTX3 smearwas further increased after NaOH treatment (FIG. 21B, lane 3 and 4),which showed that AMGnE contained more HMW PTX3 than AME. More PTX3 waspresent in water-insoluble part of AM. CHGnE only had a HMW band in theloading well but no HMW PTX3 smear regardless of treatment with orwithout NaOH. UCGnE and UJGnE had the same pattern of PTX3 with orwithout NaOH treatment as AMGnE except the intensity of PTX3 smear was alittle weaker than that in AMGnE (FIG. 21B, lane 3 and 4). The intensityof PTX3 smear in UCGnE was also lower than that in UCE, which showedthat UCGnE contained less HMW PTX3 than UCE; that is more PTX3 waspresent in the water soluble part of UC.

The above results showed that both AM and UC contained HMW PTX3. In AMmore HMW PTX3 was water-insoluble and could be extracted by GnHCl afterPBS extraction, while more HMW PTX3 in UC was water soluble that couldbe mostly extracted by PBS.

Both IαI and HC1 Mostly in AM PBS Extract but in UC Most IαI in PBSExtract While HC1 in GnHCl Extract, While More Bikunin Present in UCGnEThan in UCE With no Difference in Others’ Two extracts

FIG. 22 shows that an 80 kDa HC1 band was present in all PBS extractsexcept UCE and in all GnE extracts except AMGnE. This band was increasedby NaOH in all PBS extracts but not in GnE, showing that AM containedboth free and bound water soluble HC1 (i.e. ester bound to HA in HC-HAand to bikunin in IαI) that was released by NaOH in agreement with Zhanget al. (2012) J. Biol. Chem. 287:12433-12444. UC contained water solublebounded HCl that was released by NaOH, and also contained waterinsoluble free HCl bound to water insoluble extracellular componentsthat were dissociated by SDS and 2-ME but not affected by NaOH. This wasconsistent with strong positive HC1 staining in UC. The HMW HC1 band waspresent in all PBS and GnHCl extracts in loading wells and was decreasedby NaOH, showing that it was the HC-HA complex. The HMW HC1 band wasweaker in UCGnE than in UCJGnE, illustrating that Wharton’s jellycontained more water insoluble HC-HA complex. Free IαI was found in allPBS extracts but not in all GnE extracts, suggesting that it was watersoluble. However, free PαI was found in all PBS and GnHCl extracts,suggesting PαI had a different interaction with IαI. More bikunin wasfound in UCGnE than in UCE with no difference in other two extracts,highlighting that most bikunin was bound to other water insolublemolecules in UC and that this was indicative of a unique function.

TSG-6 Was Present in the HMW Complex of AM but Not UC GnHCl Extract

FIG. 23 showed that the 35 kDa TSG-6 band, which has been reported inAME (Zhang et al. (2012) J. Biol. Chem. 287:12433-12444), was present inAMGnE but not all other extracts, showing that TSG-6 was absent in GnEof UC. This band was not affected by NaOH, confirming that TSG-6 was notbound to HMW species that can be cleaved by NaOH. However, the HMW TSG-6band was found in AMGnE and CHGnE, but not in UCGnE and UJGnE.Furthermore, this band was not changed by NaOH, showing that TSG-6 wasstrongly bound to HMW species. TSG-6 was not detected in HC-HA purifiedby 4X ultracentrifugation from GnE, suggesting that although TSG-6 isstill bound in the insoluble matrix, it is separable by GnHCl duringultracentrifugation.

In summary, GnHCl further extracted abundant HA and proteins from theinsoluble part of AM and UC after PBS extract. UC contained more HA thanAM and CH in both PBS and GnHCl extracts. HMW PTX3 were at a higherlevel in AM GnHCl extract and at a higher level in UC PBS extract. MoreHMW PTX3 was retained in the insoluble part after PBS extract. HC1 wasmostly in AM PBS extract but not in UC GnHCl extract. HMW TSG-6 was inAMGnE but not in UCGnE and UJGnE, showing that TSG-6 was still bound inthe insoluble matrix but separable by GnHCl during ultracentrifugation.

Example 28: Purification of the HC-HA Complex by Four SuccessiveUltracentrifugations from AM and UC PBS Extract and Detection of thePresence of PTX3, HCs, Bikunin, and TSG-6 in the HC-HA Complexes

In this example, HC-HA complex was purified by four successiveultracentrifugations from AME and UCE, and the presence of PTX3 as wellas HCs, bikunin and TSG-6 were detected in the AM and UC HC-HA complexby Western blot.

The 4^(th) AM HC-HA Complex Contained More HMW PTX3 and HC1, and wasPurer Than the 2^(nd) and 3^(rd) HC-HA Complex

With the anti-PTX3 antibody, Western blot analysis of 1-4^(th) AM HC-HAshowed a 90 kDa band (dimer), compared to the monomer which was found inPBS extracts. This showed that the dimer status was resolved by furtherextraction in 4 M GnHCl through ultracentrifugation, revealing and a HMWband in gel top in 1st, 2nd, 3rd and 4^(th) HC-HA complex (FIG. 24 a ).Compared with purified PTX3 control, the 90 kDa band was PTX3 dimer, andthe high molecular weight band was PTX3-containing HC-HA complex. AfterHAase treatment, the 90 kDa band did not change in all HC-HA complexes,but the HMW smear band was vaguely detected in 3^(rd) and 4^(th)fractions. That is, from 1^(st) to 4^(th), the HMW band graduallydisappeared and a smear gradually appeared which was more intensified in4^(th) HC-HA complex. There was no 45 kDa PTX3 monomer band in all HC-HAcomplexes. The results showed that HC-HA complex contained multimericform of PTX3 that is able to bind to HC-HA, and with the number of timesof ultracentrifugation increasing, the PTX3-containing HC-HA complexbecame more purified. The existence of 90 kDa PTX3 dimer in HC-HAcomplex with or without HAase showed that: 1) PTX3 dimer present inHC-HA was dissociated by SDS and 2-ME, and 2) the HMW PTX3 was resistantto SDS and 2-ME. The PTX3 dimer being a product due to 2ME is furtherconfirmed below (FIG. 24 a ).

With anti-HC1 antibody, an 80 kDa HC1 band was detected only in earlier1^(st) and 2^(nd) fractions from all the four HC-HA complexes (FIG. 24 b). After HAase treatment, the HC1 band intensified, and several smallerbands also appeared in 1^(st) to 3^(rd) HC-HA complexes. The resultsshowed that purified HC-HA complex did not contain free HC, and HC-HAwas made of HC1. In agreement with above PTX3 Western blot results, withincreasing times of ultracentrifugation, the HC-HA complex became morepurified. No HC2 (FIG. 24 c ), HC3, and TSG-6 (FIG. 24 d ) were found inall HC-HA complexes.

TSP-1 Was Only Present in AM GnHCl Extract but Not in PBS Extract and1-4^(th) HC-HA Complex

Thrombospondin-1 (TSP-1) was only detected as trimer in AM GnHCl extractbut not in PBS extract and 1-4^(th) HC-HA complex (FIG. 25 ). AfterHAase treatment it appeared as a smear, illustrating that TSP-1 waswater insoluble and strongly bound to HC-HA. However, such a binding inthe insoluble matrix to HC-HA can be dissociated by GnHCl and CsCl.

Like 4^(th) AM HC-HA Complex, 4^(th) UC HC-HA Complex Contained PTX3 andHC1 but not HC2, HC3, Bikunin and TSG-6 and the Lack of 2ME did notGenerate PTX3 Dimer but Yielded HC1

With the anti-PTX3 antibody, Western blot analysis of 4^(th) UC HC-HAwith or without HAase treatment showed a similar PTX3 band pattern in4^(th) UC HC-HA complex (FIG. 26 a , lanes 5 and 6) to that in 4^(th) AMHC-HA complex (FIG. 26 a , lanes 3 and 4). When the sample buffer didnot contain 2-ME, the 90 kDa PTX3 dimer disappeared in UC HC-HA complexwith or without HAase (FIG. 26 a , lanes 7 and 8), showing that theappearance of 90 kDa PTX3 dimer in HC-HA complex was due to thereduction of PTX3 bound to HC-HA by 2-ME in the sample buffer. Withanti-HC1 antibody, only a high molecular weight HC-HA band was detectedin 4^(th) UC HC-HA complex (FIG. 26 b , lane 6) similar to that in4^(th) AM HC-HA complex (FIG. 26 b , lane 4). After HAase the HC-HA banddisappeared and the HC1 band increased (lane 7) like 4^(th) AM HC-HAcomplex (lane 5), though its intensity was a little weaker than that in4^(th) AM HC-HA. This showed that HC1 formed a complex with S-S withPTX3. The stronger band with a MW slightly higher than generic HC1appeared when the sample buffer did not contain 2-ME, showing that HC1was linked to PTX3 via S-S. No HC2, HC3 (FIG. 26 c ), bikunin (FIG. 26 d) and TSG-6 (FIG. 26 e ) was detected in 4^(th) AM and UC HC-HA complex.

Example 29: Purification of HC-HA Complex From Total AM and UC GnHClExtract by Four Successive Ultracentrifugations and Comparison with PBSExtractions

This example determined that more HC-HA complex from AM and UC could beobtained, the HC-HA complex had a more reasonable constitution of PTX3and HC-HA, and that it had more effective therapeutic role in theclinic. AM and UC was extracted by 6M GnHCl buffer (200 mM Tris-HCl, pH8.0, 6M GnHCl, 10 mM EDTA, 10 mM aminocaproic acid,10 mMN-ethylmaleimide, 2 mM PMSF): GnHCl extraction from AM and UC wasperformed by adding 6 M GnHCl buffer to AM and UC powder at 1:4 (g/ml).Samples were mixed overnight at 4° C., and centrifuged at 48,000 g, at4° C. for 30 min. The supernatants were GnHCl extracts. The 4^(th) HC-HAcomplex was purified from AM and UC GnHCl extract using the sameprocedure as that for the 4^(th) HC-HA purification from PBS extract.Characterization of HC-HA complex was performed by Western blotting toexamine PTX3, HCs, bikunin, TSG-6 and likely other proteins. An agarosegel of HC-HA was run to see HA content and molecular weight.

GnHCl Extracted More HC-HA Complex From AM and UC Than PBS

The GnHCl extracts from AM and UC were named AMEG and UCEG, and their HAand protein contents were detected by BCA assay and HA ELISA,respectively. 4^(th) HC-HA complex was purified from GnHCl extract andtheir HA and protein contents were similarly detected. Table 2summarizes the contents of protein and HA in both PBS and GnHCl extractsand their 4^(th) HC-HA complex. The results showed that AM and UC GnHClextracts contained more HA and have a higher HA/protein ratio comparedto relative PBS extract. Furthermore, more HC-HA complex was purifiedfrom GnHCl extract.

TABLE 2 Quantitation of proteins and HA in extracts and 4^(th) HC-HAfrom AM and UC. Sample Tissue weight/Extract buffer volume (g / ml) HA(µg/ml) Protein (µg/ml) HA/protein Ratio (µg/µg) Relative 4^(th) HC-HAHA (µg/ml) Protein (µg/ml) AME-PBS J021 1:1 61.5 5353.0 0.011 4undetectable AMEG G021 1:4 431.4 3762.5 0.115 11 undetectable UCE-PBSI103 1:1.5 453.5 8523.8 0.053 32 89.1 UCEG G021 1:4 442.0 4750.0 0.09344 13 UCmixE-PBX 1:4 277.5 5135.1 0.054 20 undetectable UCmixEG 1:6441.6 4730.0 0.093 40 undetectable

AM 4^(th) GnHCl HC-HA complex contained more HC1 and HMW PTX3 butcontained less than the PBS HC-HA with or without HAase or NaOHtreatment, and both PBS and GnHCl HC-HA did not contain TSP-1.

With the anti-HC1 antibody, GnHCl HC-HA showed a HMW band in the loadingwell as PBS HC-HA, but HAase digestion only released weaker HC1 (FIG. 27a , lanes 6 and 7). NaOH treatment also released a weaker HC1 band thathad a little higher MW than that released by HAase (FIG. 27 a , lane 8),which was not seen in PBS HC-HA after NaOH treatment. These resultsshowed that GnHCl HC-HA contained HC1 but the amount was less than PBSHC-HA. Similarly, not like PBS HC-HA, with anti-PTX3 antibody, GnHClHC-HA only showed dimer PTX3 but no notable HMW PTX3 smear with orwithout HAase digestion (FIG. 27 b , lanes 6 and 7). NaOH also resultedin a HMW and dimer PTX3 appearance. These results showed that GnHClHC-HA contained less HMW PTX3 than PBS HC-HA.

Similar to HC1 blot, a higher MW dimer of PTX3 occurred after NaOH thanHAase in GnHCl HC-HA. These results collectively indicated that NaOHreleased an ester bond that links HC1 to HA and could be associated withPTX3. Because GnHCl HC-HA had more HA content than PBS HC-HA, the GnHClHC-HA complex contained HA that was not bounded by PTX3 or HC1 resultingin the decrease of actual HC-HA/PTX3 complex content in the purifiedproducts and less HC1 and HMW PTX3 in it. TSP-1 was not detected in PBSHC-HA with anti-TSP-1. It should be noted that TSP-1 was also notdetected in GnHCl HC-HA. Because GnHCl extract contains TSP-1, theseresults showed that TSP-1 dissociated by ultracentrifugation, so it wasnot present in GnHCl HC-HA.

Agrose Gel Showed Abundant HA in GnHCl HC-HA

PBS HC-HA showed a “continuous HA smear from the top loading well to thebottom of agarose gel, and HAase completely abolished the HA smear (FIG.28 , lanes 3 and 4). GnHCl HC-HA showed a band in the loading well and aHA smear, which started from the 4,570 kDa location to the bottom of theagarose gel (FIG. 28 , lane 5). HA in GnHCl HC-HA had a break betweenthe loading well to the beginning of the HA smear, although itsintensity was stronger than that in PBS HC-HA. Furthermore, HAase didnot completely abolish the HA smear and HMW HA band in GnHCl HC-HA (FIG.28 , lane 6). The top fractions (1-6 fractions) from GnHCl extract afterthe 4^(th) ultracentrifugation also showed the same HA smear pattern asthe “bottom fractions” of GnHCl HC-HA (FIG. 28 , lanes 7 and 8). Theseresults showed that GnHCl HC-HA contained more HMW HA (with a MW smallerthan PBS HC-HA ) but lacked a portion of HMW HA smear that correspondedto the lack of HMW PTX3 smear in the Western blot of GnHCl HC-HA. Thisindicated that the missing HMW HA smear, which was present in PBS HC-HA,was at least partly formed by crosslinking of PTX3 and HC-HA, and thatthe HMW HA in the loading well of GnHCl HC-HA was complexed withcomponents other than PTX3

GnHCl HC-HA contained some proteins that are not found in PBS HC-HA byCoomasie blue staining

FIG. 29A shows that bands in the top loading well, a major 140 kDa andsome minor 70 kDa, doublet 55 kDa and 20 kDa bands, were present in AMGnHCl HC-HA and in the top fractions of GnHCl HC-HA but not in all otherPBS HC-HA. This showed that AM GnHCl HC-HA contained some proteins thatwere absent in PBS HC-HA. In addition, a 90 kDa and 25 kDa band werealso visualized in the GnHCl HC-HA top fractions. Because Western blotdetected HC1 in PBS HC-HA, the HC1 should also be present in PBS HC-HAby Coomassie blue staining. The reason why it was not present in PBSHC-HA was due to the fact that the loaded HC-HA did not enter the geldue to over-loading. The 140 kDa showed as a broad band suggesting itcontained sugar moieties. In addition, HAase did not affect these bands,showing that these species were dissociated by SDS and 2-ME.

Compared to AM GnHCl HC-HA, UC GnHCl HC-HA showed bands in the toploading well, a 90 kDa, 70 kDa, doublet 55 kDa, 35 kDa and 20 kDa bands(FIG. 29B, lanes 4 and 5). A faint 140 kDa band was also present in it.These bands were not affected by HAase. In addition, the top fractionsof GnHCl HC-HA also showed a smear from top well to the site of 200 kDawhich decreased after HAase treatment. All the bands were absent in UCPBS HC-HA. These results showed that UC GnHCl HC-HA also contained someproteins that were absent in PBS HC-HA, and UC GnHCl HC-HA was differentfrom AM GnHCl HC-HA regarding the protein bands they contained.

The above results showed that GnHCl HC-HA was different from PBS HC-HAfrom both AM and UC in the following aspects: (1) GnHCl HC-HA containedless HC1 and HMW PTX3 than PBS HC-HA (from Western blot) while like PBSHC-HA, TSG-6, HC2 and HC3 were also not present in GnHCl HC-HA. (2)GnHCl HC-HA contained more HMW HA but lacked a piece of HMW HA thatcorresponded to the HMW PTX3 smear in Western blotting shown by PBSHC-HA (from agrose gel). (3) GnHCl HC-HA contained some proteins mainlywith MW 140 kDa that were not found in PBS HC-HA (from Coomassie bluestaining gel).

In summary, GnHCl extracted more HA and proteins from AM and UC tissue,resulting in a higher HA/protein ratio compared to PBS extract. MoreHC-HA complex (according to HA content) was purified from the GnHClextract for both AM and UC. GnHCl HC-HA contained HC1 and HMW PTX3 butmuch less than in PBS HC-HA for both AM and UC. GnHCl HC-HA lacked aspecies of HMW HA smear in the agrose gel that corresponded to the HMWPTX3 smear in Western blotting shown by PBS HC-HA. GnHCl HC-HA containedsome proteins that are not found in PBS HC-HA.

Example 30: Determination of the Identity of Unknown Protein Bands inGnHCl HC-HA Complex Purified From AM and UC

This example determined the identify of unknown bands in GnHCl HC-HA byrunning SDS-PAGE gels followed by either CB staining or Western blotanalysis of GnHCl HC-HA from AM and UC with or without deglycosylation.The sample was lyophilized AM and UC 4x HC-HA (contained 30 µg HA) fromboth PBS and GnHCl extracts. Lyophilized HC-HA were incubated with 50 µlTFMS and 20 µl anisole on ice for 3 h and neutralized with TFMS with 125µl N-ethylmorpholine. Samples were precipitated with 5-10 volumes ofacetone overnight at -20 C or for 1 h at -80 C. Samples were centrifugedand the dried pellet was dissolved in SDS sample loading buffer forelectrophoresis. Enzymatic deglycosylation with keratinase(Endo-β-galactosidase) was performed to remove keratan sulfate chain andN-linked oligosaccharides, or with Chondroitinase (Cabc) to removechondroitin sulfate chain. HC-HA (contained 30 µg HA) was incubated with0.1 U/ml keratinase in 50 mM sodium acetate, pH 5.8, at 37 C for 2 h, orincubated with 5 U/ml Cabc in PBS at 37 C for 2 h. An SDS-PAGE gel wasrun to test for CB staining, followed by Western blot analysis.

Keratan Sulfate and Osteoadherin Were Present in AM GnHCl HC-HA but notin PBS HC-HA

Western blot analysis was performed. The results are shown in FIGS. 30.Band C. Western blot with anti-keratan sulfate antibody showed a broad 70kDa (60-80 kDa) band in AM GnHCl HC-HA with or without HAase digestion(FIG. 30B, lanes 6-8), but not in PBS HC-HA, which showed that this ~70kD keratin sulfate proteoglycan was responsible for the positiveimmunostaining shown in FIG. 30D. This 70 kD band corresponded to thesame band noted in GnHCl HC-HA with or without HAase treatment shown inCoomassie Blue stained gel (FIG. 30A).

To further determine if this 70 kD keratin sulfate proteoglycan is aSLRPs, anti-lumican, anti-fibromodulin, and anti-osteoadherin antibodieswere used in the Western blot. The anti-osteoadherin antibody recognizeda 60 kD band, but not a 70 kD band in GnHCl HC-HA with or without HAasedigestion (FIG. 30C, lanes 6-8), but not in PBS HC-HA. Osteoadherin withkeratin sulfate chain has a molecular mass of ~80 kD, while itsnon-keratin sulfate protein is ~60 kD. A keratin sulfate band in thesize of 60 kD was detected, but only in a broad size of 70 kD, whichshowed that the 60 kD band detected by anti-osteoadherin was non-keratansulfate osteoadherin. AM GnHCl HC-HA contained non-keratan sulfateosteoadherin that tightly associated with HC-HA and withstood 4 timesultracentrifugations in the presence of 6M GnHCl and cesium chloride.But, it was released by SDS and 2-ME in the sample buffer. The resultsalso showed that the 70 kD keratin sulfate proteoglycan was notosteoadherin. There was no lumican and fibromodulin detected in GnHClHC-HA, which showed that the 80 kD proteoglycan is neither lumican norfibromodulin.

Deglycosylaton and Analysis of AM GnHCl HC-HA

HC-HA was deglycosylated by TFMSA to remove all glycans using keratinaseand chondroitinase to remove specific glycans to see whether there wereany changes to the 140 kD and ~80 kD bands in AM GnHCl HC-HA, andfurther determine whether the ~80 kD keratin sulfate proteoglycan wasKeratocan, PRELP or Osteoglycan. As a first step confirming the effectof the above deglycosylation, Coomassie Blue staining was performed.

In FIG. 31A, the Coomassie Blue (CB) stained gel, AM GnHCl HC-HA (FIG.31A, lane 2) showed the same bands of dominant 140 kDa, 70 kDa, doublet50 kDa, 20 kDa and a weak 35 kDa band as well as a HMW band in the topof gel. K/C/H did not greatly affect these bands except for generating amajor 80 kDa, a weak 100 kDa, and 30 kDa bands appearance (FIG. 31A,lane 3). This pattern was similar to that derived from the top fractionof AM GnHCl HC-HA under C/K/H (FIG. 31A, lane 6). These results showedthat the 140 kDa, 70 kDa and 55 kDa bands were not keratan sulfatedand/or chondroitin sulfated, and that there were other keratan sulfatedand/or chondroitin sulfated proteins in GnHCl HC-HA that were releasedas a major 80 kDa species after C/K/H. TFMSA treatment led todisappearance of all of the above bands except the 20 kDa band, andgenerated a clear new 50 kDa band and a HMW smear (FIG. 31A, lane 4).TFMSA/H made the smear disappear resulting in a new 25 kDa band but didnot change the 50 kDa band (FIG. 31A, lane 5). This result showed 140kDa, 70 kDa, 55 kDa and 80 kDa band were the same species of 50 kDa thatwith different amounts of glycan chains.

To determine whether the 50 kDa band originated from 60 kDaosteoadherin, Western blot analysis was performed with ananti-osteoadherin antibody. The result showed a 60 kDa species in AMGnHCl HC-HA (FIG. 31B, lane 4), consistent with the finding shown inFIG. 31C. C/K/H did not change its molecular mass (FIG. 31B, lanes 5 and6), but TFMSA with or without HAase (T/H) treatment completely changedit into a 55 kDa species with less intensity (FIG. 31 , lanes 7 and 8).The top fraction of AM GnHCl HC-HA with T/H showed a stronger band but asmaller MW without the intensity change compared to without T/H (FIG.31B, lanes 9 and 10). These results further confirmed that AM GnHClHC-HA contained osteoadherin, which was free of keratan sulfate andchondroitin sulfate. The reason why the intensity of osteoadherin banddecreased after TFMSA treatment was due to (1) the protein was degradedby TFMSA; (2) it was blocked by other large amount of proteins with thesame MW that were also released after TFMSA treatment. Osteoadherin wasnot detectable in PBS HC-HA without any treatment, but a doublet bandsof 60 kDa appeared after TFMSA/HAase treatment, showing that PBS HC-HAcontained a minute amount of osteoadherin, which was tightly bound toHA.

Western blot with an anti-decorin antibody showed a very strong broad140 kDa species (80-160 kDa) and a weak doublet 50 kDa species in AMGnHCl HC-HA (FIG. 31C, lane 4; FIG. 31D, lanes 4 and 5) but not in PBSHC-HA (FIG. 31C, lane 2; FIG. 31D, lanes 2 and 3). The broad 140 kDaspecies corresponded to decorin with one chondroitin sulfate or dermatansulfate chain and different number of glycans while doublet 50 kDaspecies likely corresponded to the less glycosylated decorin. BecauseHAase did not affect decorin species, it showed they can be released bySDS and 2-ME. The above notion was confirmed by C/H, which increased a70 kDa species (FIG. 31C, lane 5), and by C/H/K, which gave the sameresult (FIG. 31B, lane 6). Hence, the 70 kDa species was thechondroitin-free decorin. This 70 kDa species was a minor componentbecause the maj or broad 140 kDa species was not greatly changed byeither C/H or C/H/K. TFMSA treatment completely deleted the broad 140kDa species meanwhile gave rise to a major 43 kDa species thatcorresponded to deglycosylated decorin core protein, and minor 95 kDa,80 kDa and a weak 30 kDa species (FIG. 31C, lane 7). TFMSA/H treatmentshowed the same species pattern as TFMSA alone except that the intensityof all these species was enhanced, showing that decorin was tightlybound to HA. TFMSA/H also resulted in a release of a faint 43 kDaspecies from PBS HC-HA, showing that AM PBS HC-HA also contained aminute amount of decorin that was tightly bound to HA. The top fractionof AM GnHCl HC-HA with or without TFMSA/H showed the same pattern as thebottom fraction with the intensity stronger than the latter (FIG. 31C,lanes 9 and 10), which showed that the top fraction also containedabundant decorin.. The above results showed that the major 140 kDa, 70kDa and doublet 50 kDa species in CB staining gel are formed by decorinvia CS and mostly non-CS and non-KS.

Unlike decorin, Western blot with an anti-biglycan antibody showed a HMWsmear with one strong area at 400 kDa, and a weak 45 kDa species in AMGnHCl HC-HA with or without HAase (FIG. 31E lane 4; 2F lanes 4 and 5)but not in PBS HC-HA (FIG. 31D, lane 2; F, lanes 2 and 3). The 45 kDaspecies corresponded to a biglycan core protein, while the HMW smear wasglycosylated biglycan with two chondroitin sulfate or dermatan sulfatechains. HAase intensified the 400 kDa area with less HMW smear above 400kDa, which showed some biglycan were also bound to HC-HA. C/H or C/K/Hdid not greatly change the HMW smear and 45 kDa species, but increased a70 kDa species (FIG. 31E, lanes 5 and 6) that was likely thechondroitin-free biglycan. Because the amount of 70 kDa species was verysmall and the major HMW smear was not greatly changed by the abovetreatments, most biglycans in AM GnHCl HC-HA were not associated with CSor KS. TFMSA treatment completely deleted the HMW smear and gave rise toa major 45 kDa species that corresponded to deglycosylated decorin coreprotein, and a weak 95 kDa, 80 kDa and 30 kDa species (FIG. 31E, lane7), which suggested the existence of biglycan in AM GnHCl HC-HA. The 95kDa and 80 kDa species were partly deglycosylated biglycan, while the 30kDa species was degraded biglycan. TFMSA together with HAase treatmentshowed the same species pattern as TFMSA alone except that the intensityof all these species enhanced, which suggested that biglycan was alsotightly bound to HA. The top fraction of AM GnHCl HC-HA with or withoutTFMSA/HAase showed the same species pattern as the bottom fraction withthe intensity stronger than the latter (FIG. 31E, lanes 9 and 10), whichshowed the top fraction also contained abundant biglycan. Western blotanalysis with an anti-keratan sulfate antibody showed the presence ofthe 70 kDa keratan sulfated protein in AM GnHCl HC-HA with or withoutkeratinase or chondroitinase treatment without shifting the molecularsize (FIG. 31G), which suggested that keratinase did not completelyeliminate the keratan sulfate or the amount of KS was minute in thisspecies. Western blot with anti-PTX3 antibody showed an increased HMWPTX3 smear which was shown in AM GnHCl HC-HA with K and even more withK/H (FIG. 31H, lanes 6 and 8) compared to that with or without HAasealone digestion (FIG. 31G, lanes 4 and 5). Chondroitinase had no sucheffect, which indicated that some KS-containing species were bound toPTX3 in GnHCl HC-HA. The same results were also obtained from Westernblot analysis of UC GnHCl HC-HA with or without keratinase digestion(see below FIG. 32G). Western blots confirmed that there was noFibromodulin, Lumican, Keratocan, PRELP, Osteoglycin, epiphycan,Periostin and TSG-6 as well as Bikunin in AM GnHCl HC-HA.

In summary, AM GnHCl HC-HA contained abundant decorin and biglycan thatwere bound to HC-HA, but PBS HC-HA contained only faint decorin and nobiglycan. AM GnHCl HC-HA contained osteoadherin and keratansulfate-containing species, while PBS HC-HA did not. A very small amountof decorin and biglycan in AM GnHCl HC-HA contained chondroitin sulfatechain.

Deglycosylaton and analysis of UC GnHCl HC-HA showed abundant present ofDecorin and biglycan in UC GnHCl HC-HA but not in PBS HC-HA. Keratansulfate, osteoadherin and bikunin were also present in UC GnHCl HC-HA.

CB staining (FIG. 32A) showed the same bands of 160 kDa, 90 kDa, 70 kDa,doublet 50 kDa, 35 kDa and 20 kDa bands in the top loading well in UCGnHCl HC-HA (FIG. 32A, lane 4). C/H or C/H/K did not greatly affectthese bands except resulting in the appearance of a maj or 80 kDa and aweak 30 kDa band (FIG. 32A, lanes 5 and 6). TFMSA treatment decreasedall above bands except the 20 kDa band but increased a major 50 kDaband, a minor 80 kDa band and a HMW smear (FIG. 32A, lane 7). TFMSA/Hmade the smear and 80 kDa band disappear but resulted in a weak 25 kDaband that appeared and decreased the intensity of the newly formed 50kDa band (FIG. 32A, lane 8). These results are similar to that obtainedfrom AM GnHCl HC-HA (FIG. 31A), showing that UC GnHCl HC-HA had asimilar constitution with AM GnHCl HC-HA. The top fraction of UC GnHClHC-HA with or without TFMSA/H showed the same pattern as the bottomfraction (FIG. 32A, lanes 9 and 10), indicating they had the samecomponents as the bottom fraction. UC PBS HC-HA without deglycosylationonly showed a HMW band in the loading well and below it, as well as a 20kDa band. TFMSA/H deleted the HMW band but mainly increased a 50 kDaband beside a weak 80 kDa and a 25 kDa band, suggesting that UC PBSHC-HA contained some glycosylated protein that only was released bycomplete deglycosylation and HA degradation.

Western blot analysis with an anti-decorin antibody showed a broad 160kDa species in UC GnHCl HC-HA with or without HAase (FIG. 32B, lanes 4and 5) but not in PBS HC-HA (FIG. 32B, lanes 2 and 3), indicating thatUC GnHCl HC-HA, like AM GnHCl HC-HA, also contained decorin. Itsmolecular mass was different than that in AM GnHCl HC-HA due to adifferent level of glycosylation. HAase greatly decreased the 160 kDaspecies, showing that it was bound to HC-HA. Keratinase with or withoutHAase also decreased the intensity of 160 kDa species, indicating italso contained some KS. Notably, C with or without HAase digestion ledto the 160 kDa species and top well species disappearing, but gave riseto a strong 50 kDa and 90 kDa species as well as a HMW smear, showingthe decorin in UC GnHCl HC-HA was mainly chondroitin sulfate incomparison to that in AM GnHCl HC-HA, where less of them containedchondroitin sulfate chain. These results further confirmed that UC GnHClHC-HA contained decorin, and that decorin in UC differed from AM in (1)glycosylation, (2) type of glycosaminoglycan is attached, and (3) theoverall amount.

Western blot with anti-biglycan antibody showed a strong HMW species intop well, a HMW smear at 400 kDa area and 140 kDa area, and a 45 kDaspecies in UC GnHCl HC-HA with or without HAase (FIG. 31C, lanes 4 and5) but not in PBS HC-HA (FIG. 31C, lanes 2 and 3). HAase intensified the400 kDa area without affecting the HMW species in the well, suggestingthat some species was tightly bound to HA. Keratinase with or withoutHAase did not greatly change above species except decreasing the 45 kDaspecies (FIG. 32C, lanes 6 and 8), suggesting that the 45 kDa speciescontained KS but most others do not. Chondroitinase alone abolished theHMW species in top well and reduced the intensity of 400 kDa area, butincreased a strong broad 50 kDa species, 100 kDa species and 28 kDaspecies with smear between them (FIG. 32C, lane 7). Chondroitinase plusHAase had the same results with the whole smear more intensified and the28 kDa species disappeared (FIG. 32C, lane 9). These results suggestedthat similar to decorin, biglycan in UC GnHCl HC-HA mainly bringschondroitin sulfate chain. This finding is different from that in AMHC-HA where less was chondroitin sulfated. Also most biglycans form HMWcomplex in HC-HA with some bound to HC-HA.

Western blot with anti-bikunin antibody showed a broad 35 kDa band in UCGnHCl HC-HA with or without HAase digestion but not in PBS HC-HA (FIG.32D, lanes 4 and 5). The 35 kDa band corresponds to the MW of nativebikunin. Keratinase with or without HAase sharpened this 35 kDa bandsharp but did not change its MW (FIG. 32D, lanes 6 and 8), whilechondroitinase with or without HAase digestion changed the 35 kDabikunin into a 25 kDa core bikunin (FIG. 32D, lanes 7 and 9), furtherconfirming the existence of bikunin in UC GnHCl HC-HA, and contained CSas reported. Because a HMW smear also formed after chondroitinasetreatment, it suggested that bikunin is tightly bound to HC-HA via CS.These results were different from AM GnHCl HC-HA that did not containbikunin.

Western blot with anti-PTX3 antibody showed a increased HMW PTX3 smearin UC GnHCl HC-HA after H, K, C, and especially with K/H (FIG. 32E,lanes 6 and 8) compared to that only with or without HAase digestion(FIG. 32E, lanes 4 and 5). These results confirmed the existence of HMWPTX3 in UC GnHCl HC-HA, and its strong binding in GnHCl HC-HA.Furthermore, such a strong binding was further helped by the presence ofKS-containing species, of which the identity remains unclear. It alsoexplained that our previous data (without enzymatic digestion) mighthave under estimated the amount of HMW PTX3 in UC GnHCl HC-HA.

Western blot with anti-keratan sulfate antibody showed a HMW species inthe well and a faint 55 kD band in UC PBS HC-HA. HAase did not changethis band but made a 60 kDa band more obvious (FIG. 32F, lanes 2 and 3).However, a 140 kD band was recognized by anti-keratan sulfate antibodyin UC GnHCl HC-HA with or without HAase digestion (FIG. 32F, lanes 4 and5). Keratinase with or without HAase treatment did not delete the 140kDa band, but increased mainly a 35 kDa band and several other bandsbetween them including a 60 kDa and a 55 kDa bands seen in PBS HC-HA(FIG. 32F, lanes 6 and 8).

Chondroitinase with or without HAase treatment also did not delete the140 kDa band, but increased a 90 kDa band as well as a 60 kDa and a 55kDa bands seen in PBS HC-HA (FIG. 32F, lanes 7 and 9). Chondroitinasetreatment also resulted in a HMW smear appearance that decreased afterHAase treatment, suggesting UC GnHCl HC-HA contained abundantchondroitin sulfated proteins beside keratan sulfated proteins. It wasclear that the keratan sulfated protein in UC GnHCl HC-HA (140 kDa) hada different MW with that in AM GnHCl HC-HA (~80 kDa), and maybe it wasdue to the different amount of glycan in the chain.

With anti-osteoadherin antibody a major 60 kD band and a weak 80 kD bandwere detected in UC GnHCl HC-HA (FIG. 32G, lanes 4) but not in PBSHC-HA. These two bands were not obviously affected by Keratinase orchondroitinase or HAase. The 80 kD should be keratin sulfatedosteoadherin, while~60 kD should be non-keratin sulfated osteoadherin.The results suggested that UC GnHCl HC-HA contained both keratinsulfated and non-keratin sulfated osteoadherin.

In summary, no Fibromodulin, Lumican, Keratocan, PRELP, Osteoglycin,epiphycan, Periostin and TSG-6 were detected in UC GnHCl HC-HA. UC GnHClHC-HA contained Decorin, biglycan, Osteoadherin, keratan sulfate andBikunin. Biglycan and decorin were abundant in it, while PBS HC-HA didnot contain an abundance of these species.

Summary

We purified HC-HA complex by 4x ultracentrifugation from both PBS andGnHCl extract of AM and UC. The HC-HA purified from GnE was quitedifferent from the HC-HA purified from PBS extract in yield and chemicalconstitution (see Table 1). In quantity, HC-HA purified from GnEcontained more HA than that purified from PBS. In chemical constitution:GnHCl HC-HA contained more HMW HA (with a MW slightly smaller than PBSHC-HA ) but lacked a piece of HMW HA that corresponded to the HMW PTX3smear in Western blotting shown by PBS HC-HA (from agarose gel). With orwithout HAase digestion GnHCl HC-HA contained less HC1and HMW PTX3 thanPBS HC-HA, but after keratinase plus HAase digestion, more PTX3 wasdetected (from Western blot), suggesting the HMW PTX3 was tightly boundto keratan sulfated proteins in GnHCl HC-HA. Neither PBS HC-HA nor GnHClHC-HA contained TSG-6, HC2 and HC3. Bikunin was present in UC GnHClHC-HA but not in UC PBS HC-HA and both AM PBS and GnHCl HC-HA. AM GnHClHC-HA contained abundant Decorin, relatively more so than UC GnHClHC-HA. Both AM and UC PBS HC-HA contained a faint amount of decorin. AMand UC GnHCl HC-HA contained abundant Biglycan, expecially in UC GnHClHC-HA. No biglycan was present in PBS HC-HA. AM and UC GnHCl HC-HAcontained Osteoadherin. AM and UC PBS HC-HA did not contain Biglycan,keratan sulfate-containing species, Fibromodulin, Lumican, Keratocan,PRELP, Osteoglycin, epiphycan, Periostin, and Osteopondin, TSP-1. Therewas no Fibromodulin, Lumican, Keratocan, PRELP, Osteoglycin, epiphycan,Periostin, and Osteopondin, TSP-1, Asporin in AM and UC GnHCl HC-HA.GnHCl HC-HA contained visible protein bands mainly with MW 200 kDa, 80kDa, and 60 kDa that were not found in PBS HC-HA (from Coomassie bluestaining gel).

TABLE 3 Summary comparison of 4x HC-HA complex purified from PBS andGnHCl extract. 4^(th) PBS HC-HA 4^(th) GnHCl HC-HA Components AM UC AMUC Wet weight (g) from one placenta 23.8 ± 4.7 48.2 ± 17.2 24.6 ± 9.433.5 Total HA content (µg) 208 ± 100 2800 ± 1697 872 ± 378 6561 HC1 +++++ + + PTX3 +++ ++ ++ by keratinase ++ by keratinase TSG-6, HC2,HC3 - - - - Bikunin - - - + Decorin +/- - +++++ ++ Biglycan - - ++++ +++Keratan sulfate - -/+ ++ + Osteoadherin - - ++ + TSP-1* - - - -Osteopondin - - - - Asporin - - - - Fibromodulin, - - - -Lumican - - - - Osteoglycan - - - - Keratocan - - - - Testican - - - -Epiphycan - - - - Periostin - - - - *Note that this information is notthe same as extract, suggesting that there is TSP-1 that is dissociableby ultracentrifugation.

TABLE 4 Summary comparison of the extract of PBS and GnE sequentiallyobtained from AM and UC. PBS extract GnHCl extract AM UC AM UC HAcontent (µg/ml) < < HC1 > < PTX3 < > TSG-6 + unknown + - TSP-1 - - + +

Example 31: Constitutive Expression of PTX3 by Human Amniotic MembraneStromal Cells Leads to HC-HA/PTX3 Complex Formation

In this example, PTX3 expression in HC-HA purified from AM and itseffect on HC-HA/PTX3 complex formation in AM was examined.

Experimental Procedures 1. Materials

Guanidine hydrochloride, cesium chloride, EDTA, anhydrous alcohol,potassium acetate, sodium acetate, sodium chloride, sodium hydroxide,Tris base, Triton X-100, 3-(N,N-Dimethyl palmityl ammonio)propanesulfonate (Zwittergent³⁻¹⁶), protease inhibitor mixture(including 4-(2-aminoethyl)- benzenesulfonyl fluoride hydrochloride,aprotinin, bestatin hydrochloride, E-64, leupeptin, and pepstatin A) andphenylmethanesulfonyl fluoride were obtained from Sigma-Aldrich (St.Louis, MO). Streptomyces hyaluronidase (HAase) and biotinylatedHA-binding protein (HABP) were from Seikagaku Biobusiness Corporation(Tokyo, Japan). Dulbecco’s modified Eagle’s medium, Ham’s F12 nutrientmixture, fetal bovine serum, Hank’s balanced salt solution, gentamicin,amphotericin B and RIPA buffer were purchased from Invitrogen (GrandIsland, NY). Slide-A-Lyzer Dialysis Cassettes (3.5 K MWCO) was fromFisher Scientific (Pittsburgh, PA). BCA Protein Assay Kit was fromPierce (Rockford, IL). HA Quantitative Test Kit was from Corgenix(Westminster, CO). 4-15% gradient acrylamide ready gels andnitrocellulose membranes were from Bio-Rad (Hercules, CA). IαI wasprepared in our laboratory from human plasma, according to the publishedmethod (1,38). PTX3 mAb (MNB4) and pAb were from Enzo Life Sciences,Inc. (Plymouth, PA). Recombinant human TNF, recombinant human Pentraxin3/TSG-14 and human/mouse TSG-6 MAb (MAB2104) were from R&D Systems(Minneapolis, MN). Mouse anti-human ITIH1 polyclonal antibody againstfull length ITIH1 and rabbit anti-human ITIH2 polyclonal antibodyagainst amino acids 124-321 were from Abcam Inc. (Cambridge, MA).HiPerFect Transfection Reagent and RNeasy Mini RNA isolation Kit wasfrom QIAGEN (Valencia, CA). Small interfering RNA (siRNA)oligonucleotides for targeting endogenous human PTX3(ACACUUGAGACUAAUGAAAGAGAGA) and non-targeting siRNA controloligonucleotides (scrambled RNA) siRNA were from OriGene Technologies,Inc (Rockville, MD). Western Lighting™ Chemiluminesence Reagent was fromPerkinElmer, Inc. (Waltham, MA). The ultracentrifuge (LM8 model, SW41rotor) was from Beckman Coulter, Inc. (Fullerton, CA).

2. Cell Cultures and Agarose Overlay

Human tissue was handled according to the Declaration of Helsinki. Thefresh human placenta was obtained from healthy mothers after electivecesarean deliveries in the Baptist Hospital (Miami, FL) via an approval(Protocol Number 03-028) by the Baptist Health South FloridaInstitutional Review Board. Primary human AM epithelial and stromalcells (designated as AMEC and AMSC, respectively) were isolated fromfresh placenta as previously described (Chen et al. (2007) Stem Cells.25: 1995-2005; Li et at. (2008) J. Cell. Physiol. 215:657-664) andcultured in supplemental hormonal epithelial medium (SHEM, whichconsisted of DMEM/F12 (1:1, v/v), 5% (v/v) FBS, 0.5% (v/v) dimethylsulfoxide, 2 ng/ml EGF, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/mlsodium selenite, 0.5 µg/ml hydrocortisone, 0.1 nM cholera toxin, 50µg/ml gentamicin, 1.25 µg/ml amphotericin B) (Chen et al. (2007) StemCells 25:1995-2005; Chen et al. (2011) Tissue Eng Part C Methods17:537-548) under a humidified atmosphere of 5% CO₂ at 37° C. Theculture medium was changed every 2 days. Cells at 80% confluence wereswitched to DMEM/F12 containing 0.5% FBS for 48 h to let the cellsbecome quiescent and then treated with 20 ng/ml TNF or 20 ng/ml IL-1βfor 4 h or 24 before subject to RT-PCR and Western blot analysis. Foragarose overlay culture, AMEC, AMSC and HSF are seed in 12-well (1×10⁵cells/well) and 6-well (2×10⁵ cells/well) plate at a density of 2×10⁴/cm² in SHEM. The medium are changed at day 1 to serum-free SHEMcontaining 5% KnockOut serum replacement and 1 mM 2-phospho-L-ascorbicacid and incubated for another 2 days. After removal of the medium, 3%agarose (low melting-type, Type VII, Sigma, A9045) in DMEM/F12 with 1 mM2-phospho-L-ascorbic acid was over-layered at 1 ml or 0.5 ml to achievea 1 mm thick gel layer at room temperature for 5-10 min before adding 3ml or 1.5 ml of serum-free SHEM media with or without 5 ng/ml TNF per 6or 12 well plate, respectively. Cells were harvested without interveningmedia changes on Days 5.

3. siRNA Transfection

AMEC and AMSC were cultured in SHEM in six-well plates till 80%confluence. Cells were switched to DMEM/F12 with 0.5% FBS for 48 h andwere transfected with PepMute™ siRNA Transfection Reagent with 100 nM ofPTX3 siRNA or scrambled (sc) RNA. After 48 h, cells were harvested andsubjected to RT-PCR and Western blot analysis.

4. Purification of HC-HA Complex From AM and Serum-Free Cultures byUltracentrifugation

HC-HA complex was purified from AM and cell cultures as previouslydescribed (He et al. (2009) J. Biol. Chem. 284: 20136-20146; Yoneda etal. (1990) J. Biol. Chem. 265:5247-5257; He et al. (2008) Invest.Ophthalmol. Vis. Sci. 49:4468-447532). In brief, cryopreserved human AM,obtained from Bio-tissue, Inc. (Miami, FL), was sliced into smallpieces, frozen in liquid nitrogen, and ground to fine powder by aBioPulverizer. The powder was mixed with cold phosphate-buffered saline(PBS) buffer at 1:1 (g/ml). The mixture was kept at 4° C. for 1 h withgentle stirring and then centrifuged at 48,000 g for 30 min at 4° C. Thesupernatant (designated as AM extract) was then mixed with a 8 Mguanidine HCl/PBS solution (at 1:1 ratio of v/v) containing 10 mM EDTA,10 mM aminocaproic acid, 10 mM N-ethylmaleimide, and 2 mM PMSF andadjusted to a density of 1.35 g/ml (AM extract) or 1.40 g/ml (cellextract) with cesium chloride, respectively, and subjected to isopycniccentrifugation at 35,000 rpm, 15° C., for 48 h. The resultant densitygradients were fractioned into 12 tubes (1 ml/tube), in which thecontents of HA and proteins were measured using HA Quantitative Test Kitand BCA Protein Assay Kit, respectively. Fractions from the firstultracentrifugation, which contained most HA were pooled, brought to adensity of 1.40 g/ml by addition of CsCl, ultracentrifuged, andfractionated in the same manner as described above. Fractions from thesecond ultracentrifugation, which contained HA but no detectableproteins, were pooled and continue to the third and the forthultracentrifugation in a density of 1.42 g/ml by addition of CsCl.Fractions from the second and the forth ultracentrifugation weredialyzed in distill water and then precipitated twice with 3 volumes of95% (v/v) ethanol containing 1.3% (w/v) potassium acetate at 0° C. for 1h. After centrifugation at 15,000 g, the pellet was briefly dried byair, stored at -80° C. and designated as AM 2^(nd) HC-HA and 4^(th)HC-HA, respectively.

5. Immunostaining

Human fetal membrane containing AM and chorion section as well as cellcultures with or without an agarose overlay were fixed with 4%paraformaldehyde at room temperature for 15 min, permeabilized with 0.2%(v/v) Triton X-100 in PBS for 20 min. After blocking with 0.2% (w/v)bovine serum albumin in PBS for 1 h, sections were incubated withbiotinylated HABP (for HA, 5 µg/ml), anti-PTX3, anti-HC1 or anti-HC2antibodies (all diluted 1:200 in blocking solution) overnight in ahumidity chamber at 4° C. After washing with PBS, they were incubatedwith Alexa Fluor 488 Streptavidin (for HA, diluted 1: 100), orrespective secondary antibodies (i.e., Alexa Fluor 488 anti-mouse IgG,or Alexa Fluor 555-conjugated anti-rat IgG,) for 1 h at roomtemperature. Isotype-matched nonspecific IgG antibodies were used as acontrol. Alternatively, sections were treated with 50 U/mL StreptomycesHAase at 37° C. for 4 h before fixation. Nuclei were stained by Hoechst33342, and images were obtained using a Zeiss LSM700 confocal laserscanning microscope (Zeiss, Germany).

6. Real-Time PCR

Total RNA was extracted from cell cultures using RNeasy Mini RNAisolation Kit. The cDNA was reverse-transcribed from 1 µg of total RNAusing a Cloned AMW First-Strand cDNA synthesis kit with oligo(dT)primer. First-strand cDNAs were amplified by qPCR using AmpliTaq GoldFast PCR Master Mix and the specific PTX3 primers (46-48).Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression wasused to normalize the amounts of the amplified products.

7. Western Blot

Culture supernatants were collected, and cell lysates were obtained bywashing cells six times with cold PBS followed by incubating in RIPAbuffer at 4° C. for 1 h with gentle stirring and centrifugation at14,000 g for 30 min at 4° C. Protein concentrations in culturesupernatants and cell lysates were quantified with a BCA Protein AssayKit. Samples were incubated in 50 mM NaOH for 1 h at 25° C. or dissolvedin 0.1 M sodium acetate buffer (pH 6.0) and incubated at 60° C. for 1 hwith or without 20 units/ml of Streptomyces HAase. They then wereresolved by SDS-PAGE on 4-15 % (w/v) gradient acrylamide ready gelsunder denaturing and reducing conditions and transferred to anitrocellulose membrane. The membrane was then blocked with 5% (w/v)fat-free milk in 50 mM Tris-HCl, pH 7.5, buffer containing 150 mM NaCland 0.05% (v/v) Tween-20 followed by sequential incubation withdifferent primary antibodies followed by their respective HRP-conjugatedsecondary antibodies. Immunoreactive proteins were visualized by WesternLighting™ Chemiluminesence Reagent.

Results Positive PTX3 Staining in AM Epithelium and the Compact Stroma

Immunofluorescence staining using anti-human PTX3 antibody was performedon crosssections of fresh human fetal membrane, which consisted of alayer of epithelium and an avascular stroma, which can be furthersubdivided into a compact layer and a spongy layer, and the subjacentcell-rich chorion (FIG. 33 , phase). Positive PTX3 staining was found inthe apical surface of the epithelium and the compact stroma. Incontrast, PTX3 staining was markedly attenuated in the spongy stroma andthe chorion (FIG. 33 , PTX3). HAase digestion did not enhance PTX3staining in the latter, suggesting that the weak PTX3 staining in thesetwo areas was not due to the masking effect. Strong positive HAimmunostaining was found in AM stroma and relatively weak staining in AMepithelium using a biotinylated HABP (FIG. 33 , HA), while a weakstaining of HA was noted in the upper layer of the chorion subjacent tothe AM stroma but strong staining in the lower layer of the chorion.This staining disappeared when the tissue section was pre-digested byHAase (FIG. 33 , HA (+HAase)) suggesting that HA staining was specific.Immunostaining of individual HC, also revealed a positive staining in AMepithelium, stromal cells and matrix, and chorion (FIG. 33 , HC1 andHC2). These results suggested the presence of PTX3 in AM predominantlyin the compact stroma and the epithelium.

Presence of PTX3 in AM Soluble Extract and Purified HC-HA Complex

To investigate further the presence of PTX3 in AM, we first performedWestern blotting analyses of AM extract obtained by an isotonic saltbuffer before and after 50 mM NaOH treatment to cleave ester bonds.Recombinant PTX3 appeared as 45 kDa, 90 kDa, 180 kDa and HMW species(FIG. 34 , lane 2). Soluble AM extract revealed 45 kDa and a HMW speciesat the bottom of the loading well (mostly not entering the gel) (FIG. 34, lane 3). NaOH treatment did not affect the 45 kDa species, butcompletely eliminated the HMW species, resulting in a HMW smear of PTX3(FIG. 34 , lane 4). These results suggested that PTX3 was present as amonomer and a HMW complex in the AME. Because the latter could bedissociated into a HMW smear by NaOH that can cleave off the estercovalent linkage between HC-HA, the HMW PTX3-containing species in theloading well might be associated with HC-HA.

To further confirm whether PTX3 was associated with AM HC-HA complex, wepurified HC-HA complex by two and four successive ultracentrifugationsfrom the AM soluble extract as previous reported (He et al. (2009) J.Biol. Chem. 284:20136-20146.; Zhang et al. (2012) J. Biol. Chem.287:12433-12444) and performed Western blotting analyses with or withoutHAase digestion. Contrasted to the monomer found in soluble AME, a 90kDa species corresponding to the size of the native PTX3 dimer was shownin AM 2nd and 4^(th) HC-HA complex besides a HMW band at the bottom ofthe loading well (FIG. 34 , lanes 5 and 7). In addition, a HMW smear wasalso seen weak in 2^(nd) HC-HA and strong in 4^(th) HC-HA complex. AfterHAase treatment, the 90 kDa dimer remained in both HC-HA complexes, butthe HMW smear was intensified in 4^(th) HC-HA and increased in 2^(nd)HC-HA with the disappearance of HMW band in gel top (FIG. 34 , lanes 6and 8) similar to the results seen in AME. The existence of 90 kDa PTX3dimer in HC-HA complex with or without HAase was found to be caused bydissociation from HC-HA caused by 2-ME as elimination of 2-ME resultedin the absence of this 90 kDa species (not shown). These resultssuggested that HC-HA complex contained PTX3 that was bind to HC-HA toform the HC-HA/PTX3 complex despite four times of ultracentrifugation.

Western blot analysis with anti-HC1 antibody showed the presence ofHC-HA complex as a HMW species at the bottom of the loading well thatdisappeared upon HAase digestion (FIG. 34 , lanes 10-13), and thepresence of HC1 in the HC-HA complex that was released from HC-HAcomplex after HAase digestion (FIG. 34 , lanes 11 and 13). We did notdetect HC2 nor TSG-6 (not shown). These results collectively confirmedthat HC-HA purified from AM only contained HC1. We also noted a primedifference between 2^(nd) and 4^(th) HC-HA complexes, that is, a free 80kDa HC1 band was detected only in the 2nd HC-HA complex (FIG. 34 , lane11), but not 4th HC-HA complex, suggesting that the latter did notcontain free HC1. The presence of HA in 4th HC-HA were verified byagarose gel electrophoresis to display as a continuous HA smear from thetop loading well to the bottom of the gel, and that such a smearing wasresolved by HAase digestion.

Expression of PTX3 mRNA and Protein by AMEC and AMSC

We then determined that PTX3 was synthesized by AM epithelial andstromal cell cultures. We cultured these cells as reported (Chen et al.(2007) Stem Cells 25(8):1995-2005; Li et al. (2008) J CellPhysiol.215(3):657-64; Zhang et al. (2012) J. Biol. Chem. 287:12433-12444) andextracted total RNA for RT-PCR and proteins for Western blot analysis,and compared them to human skin fibroblasts (HCF), which were reportedto express PTX3 mRNA and protein only under the stimulation ofpro-inflammatory cytokine such as TNF and IL-1. As expected, qRT-PCRresults showed that expression of PTX3 transcript was low in restingHSF, but upregulated by TNF and IL-1β (FIG. 35A). Although expression ofPTX3 transcript in resting AMEC and AMSC was also low, it wasdramatically elevated by TNF or IL-1β (FIG. 35A). Western blottinganalysis confirmed that PTX3 protein was low in lysates (45 kDa) andundetectable in media in resting HSF (FIG. 35B, lane 2) but was detectedin lysates but not media after addition of TNF or IL-1β for 24 h. Incontrast, PTX3 protein was detectable in resting AMEC and notablyincreased by TNF or IL-1b in both lysates (45 kDa) and media (45 kDa and90 kDa) (FIG. 35B, lanes 5, 6 and 7), with TNF being more effective thanIL-1b. The same finding was noted in AMSC (FIG. 34B, lanes 8, 9 and 10).To verify that the 75 kDa and 135 kDa bands in lysates and the 50 kDaband in media from all cells were nonspecific because these bands didnot changes under TNF or IL-1b, PTX3 siRNA transfection was performedand indeed downregulated the 45 kDa species in lysis and both 45 kDa and90 kDa species but not these non-specific bands. (FIG. 35C). Theseresults collectively suggested that PTX3 was indeed synthesized andsecreted by resting AM cells and was further upregulated byproinflammatory stimuli.

Production of HC-HA/PTX3 Complex by AM Stromal Cells

Previous studies have shown that HC-HA (i.e. SHAP-HA) complex can beisolated from the cell layer of cultured mouse dermal fibroblasts in amedium supplemented with FBS, and that the isolated HC-HA contains bothHC1 and HC2 of IαI derived from FBS (Yoneda et al. (1990) J. Biol. Chem.265:5247-5257; Huang et al. (1993) J. Biol. Chem. 268:26725-26730).However, we reported that AM cells produce HC-HA by using theirendogenously generated IαI (Zhang et al. (2012) J. Biol. Chem.287:12433-12444). Because AM cells synthesized PTX3 protein which wasfurther increased by TNF and IL-1, we aimed to determine whether theyproduced HC-HA that also contained PTX3. We used HSF as a control, whichexpressed PTX3 only under pro-inflammatory stimuli, e.g. TNF and IL-1,in a serum-containing condition (Yoneda et al. (1990) J. Biol. Chem.265:5247-5257; Huang et al. (1993) J. Biol. Chem. 268:26725-26730) (FIG.35 ) and compared to AMECs and AMSCs cultured in both serum-free andserum-containing condition with or without TNF. We also overlayed 3%agarose over cell monolayers because this method has been found toentrap secreted procollagen at or near the cell surface, rather thaninto the media on keratocyte cultures (Hassell et al. (2008)Experimental Eye Resarch. 87:604-611; Etheredge et al. (2010) MatrixBoil. 29:519-524). After 5 days of overlay, HSF, AMSC and AMEC becamemore compact especially in the serum-containing condition (FIG. 36 ).The epithelial morphology became more distinct in AMEC.

We then determined whether 3% agarose overlay was also effective inentrapping secreted HA by measuring the HA concentration in culturemedia by HA ELISA assay. Without agarose overlay, the HA level wasreadily detectable in the serum-free medium of both AMSC and AMEC butnot in that of HSF. TNF significantly increased the HA level of allthree cell cultures (FIG. 37 ). The above pattern was further promotedin the serum-containing medium. Agarose overlay reduce the HA level morethan 50% in both serum-free and serum-containing conditions among allthree cultures. These results suggested that agarose overlay indeedreduced the HA level into culture medium.

To determine when secreted HA was indeed entrapped in the extracellularmatrix after agarose overlay, we performed double immunostaining forPTX3/HA, HC1/HA, and HC1/PTX3 with biotin-labeled HABP, specificantibody to HC1 and HC2, and two different anti-PTX3 antibodies, i.e.MNB4 and biotin-labeled pAb, respectively. In the serum-free condition,positive HA staining was noted in the pericellular region in HSF, whilePTX3 staining was negative (FIG. 38A). With TNF stimulation, positivePTX3 staining was observed in the cytoplasm (FIG. 38B), confirminginducible expression of PTX3 by TNF in HSF. TNF did not greatly increasethe HA staining intensity but induced a cable-like structure (FIG. 38G)similar to what has been reported in cultured renal proximal tubularepithelial cells (Selbi et al. (2006) Kidney International. 70:1287-1295). No colocalization of PTX3 with HA was observed, suggestingthat PTX3 was not associate with HA in HSF after TNF stimulation. Incontrast, positive HA staining was detected in resting AMSC as afibrillar network on the cell surface and extracellular matrix as wascolocalized with HA in the extracellular matrix (FIGS. 38C and 6K). TNFfurther increased PTX3 staining intensity and the amount of HA fibrils(FIGS. 38D and 38K).

In resting AMEC, positive HA staining was also found in extracellularspaces with some fibrillar appearance but only in sporadic areas wherecells were not as compact (FIGS. 38E and 38L). PTX3 and HAcolocalization was also observed in AMEC. TNF treatment furtherincreased PTX3 staining intensity and the amount of HA fibrils (FIG.38F). These results further confirmed that PTX3 was constitutivelyexpressed by AMSC and AMEC, and its expression can by further increasedby TNF. Weak positive HC1 staining was observed only in some HSF with(FIGS. 38G and 38J) or without (not shown) TNF treatment, and wascolocalized with HA especially in HA cable-like structure (FIG. 38G).However, we did not note colocalization between HC1 and PTX3 (FIG. 38J).In contrast, strong positive HC1 staining was noted in AMSC andcolocalized with HA on the cell membrane (FIG. 38H) and colocalized withPTX3 in the cytoplasm (FIG. 38K). AMEC also showed the strong positiveHC1 staining and colocalization with HA in the cell membrane (FIG. 38I)and with PTX3 in the cytoplasm and the cell membrane (FIG. 38L).Collectively, these results suggested that HA-rich matrix waseffectively trapped by agarose overlay in AMSC, AMEC and HSF, andcontained both HC1 and PTX3 only in AMSC and AMEC in the serum-freecondition, further confirming that such HC-HA/PTX3 complex wassynthesized by endogenous IαI.

To further confirm the formation of HC-HA/PTX3 complex under agaroseoverlay, we extracted cell layer with 6M GnHCl and performed Westernblot analysis in AMSC and HSF cultures under both serum-free andserum-containing condition with or without NaOH treatment. Serum-freeHSF showed a 170 kDa and a 140 kDa species but not 45 kDa speciescorresponding to the control PTX3 with or without TNF stimulation andNaOH treatment, suggesting that these two bands were not specific (FIG.38D, lane 2-5). Serum-containing HSF with TNF showed a faint 140 kDaspecies and some small MW species similar to those seen in serum-freeHSF (FIG. 39 , lanes 11 and 12). In contrast, serum-free AMSC showed aweak HMW PTX3 smear, a 90 kDa and a 45 kDa species (FIG. 39 , lane 6),of which the later two corresponded to PTX3 dimmer and monomer,respectively (FIG. 39 , lane 2). These results were similar to thoseseen in AME (FIG. 34 ). The same non-specific 170 kDa species and somesmall molecular species as seen in serum-free HSF were also observed.NaOH treatment increased the HMW PTX3 smear but did not affect the PTX3monomer and dimmer (FIG. 39 , lane 7) and other species. TNF increasedthe HMW PTX3 smear, PTX3 dimmer and monomer as well as other species(FIG. 39 , lane 8 and 9). Serum-containing AMSC with TNF showed strongHMW PTX3 smear, 90 kDa PTX3 dimmer and 45 kDa PTX3 monomer (FIG. 39 ,lane 13). NaOH treatment greatly increased the HMW PTX3 smear and twospecies at 60 kDa and 50 kDa (FIG. 39 , lane 14). Because NaOH break theester bound between HC and HA leading to dissolution of HC-HA, theseresults suggested that HMW PTX3 was released from HC-HA complex.Collectively, above results suggested that AMSC but not HSF producedHC-HA complex containing PTX3.

In Vitro Reconstitution of HC-HA/PTX3 Complex (rcHC-HA/PTX3)

To further confirm how HC-HA/PTX3 complex could be generated, we wouldlike to reconstitute HC-HA/PTX3 complex in vitro with HA, TSG-6, IαI andPTX3. We first immobilized HA on plastic, and successfully addedrecombinant TSG-6, purified IαI or serum as a source of IαI. It has beenreported that TSG-6 can form stable TSG-6/HA complex by binding to HAthat is immobilized on a solid surface (Wisniewski et al. (2005) J BiolChem. 280:14476-84), and that both free and HA-bound TSG-6 can transferHCs from IαI to immobilized HA to form HC-HA (Colon (2009) J Biol Chem.284:2320-31). Western blot using an anti-IαI antibody did not detect anyspecies in control iHA alone as expected (FIG. 40A, lane 2). When IαIand TSG-6 were simultaneously added to iHA, a weak 220 kDa IαI, 85 kDaHC2, a strong ~80 kDa HC1 and a 50 kDa species were detected (FIG. 40A,lane 3), suggesting that both HC1 and HC2 were transfer to iHA in thepresence of TSG-6 to form HC-HA. Comparison of the band intensity of HC1to that of HC2 suggested that more HC1 were transferred to iHA than HC2by TSG-6, resulting in a truncated 50 kDa species. When PTX3 weresimultaneously with IαI and TSG-6 to iHA, the intensity of IαI speciesdecreased but the HC1 intensity increased in a PTX3 dose-dependentmanner (FIG. 40A, lanes 4-6), meanwhile HC2 was not detectable,suggesting that PTX3 preferentially promoted transfer of HC1 but not HC2to immobilized HA catalyzed by TSG-6. When PTX3 were added aftersimultaneous addition of IαI and TSG-6, HC1 but not IαI or HC2 wasdetected, and the HC1 intensity was also increased in a PTX3dose-dependent manner (FIG. 40A, lanes 7-9), confirming that PTX3promoted transfer of HC1 but not HC2 to immobilized HA. These resultssuggested that PTX3 uniquely promoted transfer of predominately HC1 toimmobilized HA to form HC1-HA complex regardless whether it wassimultaneously or sequentially. Western blot using the anti-TSG-6antibody showed that both 35 kDa TSG-6 monomer and 75 kDa dimer weredetected in rcHC-HA formed by simultaneous or sequential addition ofPTX3 in midst of IαI and TSG-6 on iHA. Because the intensity of theTSG-6 band decreased in a PTX3 dose-dependent, suggesting that TSG-6bound to immobilized HA could be competed out by PTX3 (FIG. 40B). TheWestern blot using the anti-PTX3 antibody showed a prominent HMW PTX3smear in the loading well with faint tetramer and dimer bands when PTX3was added simultaneously with IαI and TSG-6, and that their intensityincreased in a PTX3 dose-dependent manner (FIG. 40C, lanes 4-6). Thisfinding suggested that PTX3 was preferentially and strongly bound torcHC-HA complex in which the binding was resistant to 8 M GnHCl wash. Incontrast, when PTX3 was added sequentially after addition of IαI andTSG-6, we did not detect HMW PTX3 and tetramer and dimer species (FIG.40C, lanes 7-9), suggesting that the binding between PTX3 and preformedrcHC-HA PTX3 was not strong to withstand 8 M GnHCl wash. Hence, these invitro reconstitution experiments suggested that PTX3 must have beenproduced simultaneously with HA, IαI and TSG-6 in vivo to allowformation of HC-HA/PTX3. This interpretation was supported byimmunocolocalization of HA, HC, and PTX3 in in vivo tissue sections aswell as extracellular matrix formed by AMSCs.

Example 33: Effects of HC-HA Complexes on TGFβ1Signaling

Immobilized HC-HA inhibits TGFβ1 signaling by downregulating expressionof TGF-β1 but upregulating TGF-β3 signaling. Such inhibition of TGFβ1signaling can withstand the challenge by addition of serum or exogenousTGF-β1 because of further suppression of TGFβRII and TGFβRIII.Consequently, immobilized HC-HA prevents corneal fibroblasts frommyofibroblast differentiation by inhibiting SMAD⅔ signaling andexpression of alpha smooth muscle formation. This action is also potentenough to revert corneal fibroblasts to keratocytes. HC-HA(Insoluble(I)) differs from HC-HA(Soluble (S)) in consisting of additional smallleucine rich proteins (SLRPs), and activates expression of TGFβ1 and BMPsignaling by upregulating expression of BMPs and their receptors, thusactivating pSMAD1/5/8, which together further promotes formation ofaggregation. These actions can further de-differentiate cornealfibroblasts to neural crest progenitors.

In this example the effect of immobilized soluble and insoluble HC-HA onTGF-β signaling in human corneal fibroblasts with or without exogenousTGFβ1 challenge was examined. In addition the effect of HC-HA complexeson SMAD⅔ signaling and suppression of αSMA expression was tested.

Experimental and clinical studies support an anti-scarring therapeuticaction by cryopreserved amniotic membrane (AM). Our studies demonstratedthat heavy chain-hyaluronan complex (HC-HA) is uniquely produced by andcan be purified from AM and suppresses the TGF-β1 promoter activity inhuman corneal fibroblasts. It is unclear whether HC-HA suppresses TGF-β1mRNA and protein expression and promotes TGF-β3 mRNA and proteinexpression known to counteract TGF-β1 signaling, and if so, whether suchinhibition of TGFβ signaling by HC-HA is translated in to suppression ofnuclear translocation of pSMAD⅔.

Mouse keratocytes can maintain an undifferentiated state (expressingkeratocan) in serum-free DMEM/ITS on plastic or in DMEM/10% FBS ifcultured on AM. Treatment of keratocytes in serum-free DMEM/ITS with 10%serum or 10 ng/ml TGF-β1induces Smad⅔ phosphorylation and nuclearlocalization (3 h) and α-SMA expression (5 days). The activation ofSmad⅔ and α-SMA in keratocytes on AM with either serum or TGF-β1treatment is suppressed (Kawakita et al. (2005) J Biol Chem.280(29):27085-92). Our studies demonstrated HC-HA suppresses the TGF-β1promoter activity in human corneal fibroblasts. It was expected thatpSmad⅔ signaling and α-SMA formation will be suppressed by HC-HA.

Human corneal fibroblasts (or Limbal niche cells, p3) were seeded onplastic dishes with or without immobilized HA, soluble HC-HA (PBS) (2Xor 4X) or insoluble HC-HA (GnHCl) (2X or 4X) for 48 h as describedabove. The cells were then treated with or without TGFβ1 for 24 h beforebeing harvested for mRNA quantitation and determination of SMAD⅔signaling. For determination of protein of TGFβ receptors, the cellswere treated with or without TGF-β1 for 48 h before collection ofprotein samples to allow time for protein expression from the expressedmRNA. For TGF-β1 ELISA, the cells were treated with or without TGF-β1for 24 h, and then cultured in the fresh medium for 24 (and 48) h. Thesupernatants were collected for TGF -β1 ELISA. For TGF-β2 and TGF-β3ELISA, the cells were treated with or without TGF-β1 for 48 h. Thesupernatants were collected for TGF-β2 and TGF-β3 ELISA. Phase contrastimages were taken up to 72 h for various cultures.

The following experiments were performed:

1. mRNA semiquantitation for TGF-β1, TGF-β2, TGF-β3 and their receptorsby real-time PCR: used for estimation of mRNA transcript expression ofTGF-β family and their receptors. Real-time RT-PCR profile consisted of10 minutes of initial activation at 95° C., followed by 40 cycles of 15seconds denaturation at 95° C., and 1 minute annealing and extension at60° C.

2. Determination of α-smooth muscle formation and SMAD⅔ signaling byimmunostaining: performed to monitor α-smooth muscle formation and SMAD⅔signaling using standard immunostaining procedure.

The experimental groups for experiments 1 and 2 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 2X HC-HA PBS 2X HC-HA PBS+TGF-β1 4X HC-HAPBS 4X HC-HA PBS+TGF-β1 2X HC-HA Gn 2X HC-HA Gn+TGF-β1 4X HC-HA Gn 4XHC-HA Gn+TGF-β1

3. Quantitation of TGFβRs by Western blotting: used to quantitateprotein concentration of TGFβRI, TGFβRII, and TGFβRIII using theircorresponding antibodies (R&D Systems). The loading sequence was asfollows:

MW marker Plastic HA 4X HC-HA PBS 4X HC-HA Gn Plastic + TGF-β1 HA +TGF-β1 4X HC-HA PBS + TGF-β1 4X HC-HA Gn + TGF-β1

4. ELISA for Quantitation of TGFβs in the Medium: The Quantikine HumanTGF-β1 and TGF-β2 ELISA Kits from R&D Systems and TGF-β3 ELISA Kit fromNorvus Biologicals are solid phase ELISAs designed to measure TGF-β1,TGF-β2 and TGF-β13 in acid activated cell culture supernatants, serum,plasma, and urine. They contain recombinant human TGF-β1, TGF-β2 andTGF-β3 and have been shown to quantitate the recombinant factorsaccurately. Results obtained using natural TGF-β1, TGF-β2 TGF-β3 showedlinear curves that were parallel to the standard curves obtained usingthe recombinant kit standards. These kits were used to determine TGF-β1,TGF-β2 and TGF-β3 in the medium. The experimental groups for experiment4 were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS +TGF-β1 4X HC-HAGn 4X HC-HA Gn+TGF-β1

Results

HCF seeded in DMEM/10%FBS formed aggregates by 72 h only on insolubleHC-HA (FIG. 41 ). Such aggregation persisted after 24 h serum starvationin DMEM/ITS (insulin/transferrin/selenium) medium. The cells were thencultivated into three different culture media: A - DMEM/ITS for 48 h;B - 24 h DMEM/ITS, 24 h DMEM/10% FBS; C- 24 DMEM/ITS, 24 h DMEM/ITSwith10 ng/ml TGF-β1. The cells cultured in HC-HA form small aggregateson 4X HC-HA (Gn), but not in other culture conditions (FIG. 41 ).However, after seeding for 2 hours in DMEM/10% FBS on immobilizedsubstrates, only HCF on the control attached well. Cells on HA, HC-HA[HC-HA (4X, PBS) and 4X, GnHCl] were all rounded, suggesting they werenot well attached. After incubation for 72 hours, all cells wereattached well, however, there were much less cells on immobilizedHC-HAs. The number of cells on these substrates was greater on HC-HA(4X, PBS) than on HC-HA (4X, GnHCl). HCF started to form aggregates onHC-HA (Gn) after 24 h of culture and condensed to bigger aggregatesafter 72 h of culture. After stimulation of TGFβ1, we observedaggregation of HCF cultured on HC-HA (4X, PBS; 4X, Gn).

In summary, HC-HA (Gn) promotes aggregation of HCF with/withoutchallenge of TGFβ1 while HC-HA (PBS) promotes aggregation of HCF underchallenge of TGFβ1. The significance of aggregation is unknown.

In DMEM/ITS, expression of, TGFβ1 and TGFβ3 transcripts were elevated byHC-HA (Gn), but TGFβ3 transcript was elevated by HC-HA (PBS) (FIG. 42 ).As expected in auto-induction, TGFβ1 and TGFβ3 mRNA were increased by 4-and 2-fold, respectively, in HCF cultured on plastic by TGFβ1 challenge,with corresponding increase of TGFβ1 protein from 60 pg/ml to 105 pg/ml(TGFβ3 protein was not detected in the experiment due experimentalerror). Under serum-free conditions, soluble 4X HC-HA reducedTGFβ1protein expression. Insoluble HC-HA also decreased secreted TGFβ1despite its promotion of TGFβ1 mRNA expression although still higherthan the control cultured in DMEM/ITS. In addition, notable suppressionof TGFβRII and TGFβRIII by both soluble and insoluble HC-HA wasobserved. Consequently, autocrine TGFβ signaling was suppressed in HCFcultured on either soluble or insoluble HC-HA but paracrine TGFβsignaling is preserved in HCF cultured on insoluble HC-HA. Furthermore,both soluble and insoluble 4X HC-HA upregulated TGFβ3 mRNA expression by3-fold under serum-free conditions without TGFβ1 stimulation, which isknown to counteract TGF-β1 signaling. Under stimulation by TGFβ1, TGFβ3mRNA expression is increased by 5- and 8-fold when HCF were cultured onsoluble and insoluble HC-HA respectively, indicating that soluble andinsoluble HC-HA strongly promotes TGFβ3 transcript expression. Theseresults also indicate that HC-HA purified from AM promotes AM’santi-scarring action by not only suppressing TGFβ1 signaling but also bymarked upregulation of TGFβ3 expression. From our results, it appearsthat HC-HA (PBS and Gn) does not affect TGFβ2 expression at both mRNAand protein levels. In summary, HC-HA (PBS) inhibits TGFβ1, butactivates TGFβ3 signaling in HCF challenged with TGFβ1 while HC-HA (Gn)activates both TGFβ1 and TGFβ3 signaling.

In the plastic control, TGFβRII mRNA was upregulated by 8-fold underTGFβ challenge (FIG. 43 ). TGFβII and TGFβIII receptor mRNA wasupregulated by HC-HA (PBS and Gn) by 2- to 8-fold respectively inserum-free condition, but completely inhibited under TGFβ1 challenge.The same result is noted for HA as well. Corresponding proteinexpression of TGFβRIIand TGFβRIII was downregulated by 3- and 3-, and 2-and 3-fold respectively when HCF cultured on HC-HA (PBS and Gnrespectively) were challenged by TGFβ. Under this situation, theseprotein expression was not changed by HA. Such downregulation maypartially explain the mechanism of the antiinflammatory and anti-scaringeffect by AM. In summary, mRNA expression of TGFBR2 and TGFβR3 wasincreased when HCF were cultured on HA and HC-HA (PBS and Gn).

Immunostaining indicated that HC-HA (PBS and Gn) inhibited pSMAD⅔nuclear translocation in both DMEM/ITS with and without TGFβ1challenge(FIG. 44 ). Such an effect was more apparent with TGFβ. This findingfurther confirmed that suppression of TGFβ1, TGFβRII and TGFβRIII wastranslated into suppression of SMAD-mediated signaling.

In addition, immunostaining results indicate that both soluble andinsoluble HC-HAs inhibited α-SMA formation after challenge of TGFβ1,further supporting that such inhibition of TGF-β1 signaling by solubleand insoluble HC-HA inhibits differentiation of HCF into myofibroblastwith or without addition of TGFβ1 (FIG. 45 ).

In summary, soluble HC-HA downregulates TGF-β1 but upregulates TGF-β3expression, while insoluble HC-HA upregulates both TGF-β1 and TGF-β3expression in HCF under serum-free and TGFβ1 challenging conditions.Because both soluble and insoluble HC-HA downregulated expression ofboth TGFβRII and TGFβRIII, these changes resulted in inhibition of TGFβsignaling as evidenced by the lack of nuclear translocation of pSMAD⅔and suppression of alpha smooth muscle formation.

Example 34: Effects of HC-HA Complexes on BMP Signaling

In this example, the effect of immobilized HC-HA with additional TGF-β1on BMP signaling was examined. The activation of BMP signaling viaactivation of pSMAD1/5/8 also was determined.

BMPs constitute a subgroup of TGFβ superfamily including BMP1-3, BMP3b,BMP4-7, BMP8a, BMP8b, BMP9-15. BMP binds type II receptors (ALK2, ALK3,or ALK6), which activates type I receptor to phosphorylates Smad1,Smad5, and Smad8, resulting in nuclear translocation of pSmad1/5/8(reviewed in Massague 2000; Herpin, 2007). It was not clear whichspecific BMPs and BMP receptors are present in HCF, and if so, whetherBMP signaling can be activated by HC-HA (PBS and Gn) and additionalTGFβ1 when the TGFβ signaling is suppression, and if so, which forms ofBMPs and BMP receptors play a major role in controlling BMP signalingand whether such an activation of BMP signaling is via pSMAD1/5/8.

Human corneal fibroblasts were seeded on plastic with or withoutimmobilized HA or HC-HA PBS (4X) or HC-HA Gn (4X) for 48 h, and thentreated with or without TGFβ1 for 24 h for mRNA quantitation anddetermination of pSMAD1/5/8 as described above. For determination ofprotein of BMP receptors, the cells were treated with or without TGF-β1for 48 h before collection of protein samples to allow for proteinexpression. For BMP ELISA, the cells were treated with or without TGF-β1for 48 h. The supernatants were collected for BMP ELISA. Phase contrastimages were taken up to 72 h for various cultures.

The following experiments were performed on the cultures:

1. mRNA semiquantitation for BMPs and their receptors by real-time PCR:used for estimation of mRNA transcript expression of BMP family andtheir receptors.

2. Determination of α-smooth muscle formation and SMAD1/5/8 signaling byimmunostaining: performed to monitor α-smooth muscle formation and SMAD⅔signaling by immunostaining.

The experimental groups for experiment 1 and 2 are:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS +TGF-β1 4X HC-HAGn 4X HC-HA Gn+TGF-β1

3. Quantitation of BMPRs by Western blotting using BMPR1A, BMPR1B andBMPR2 antibodies: used to quantitate protein concentration of BMPR1A,BMPR1B and BMPR2, respectively. The loading sequence was as follows:

MW marker Plastic HA 4X HC-HA PBS 4X HC-HA Gn Plastic+ TGF-β1 HA+ TGF-β14X HC-HA PBS + TGF-β1 4X HC-HA Gn+ TGF-β1

4. ELISA for Quantitation of BMPs in the Medium: We used BMP ELISA kits(R&D Systems) to determine BMPs in the medium. The experimental groupsfor experiment 4 are:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS +TGF-β1 4X HC-HAGn 4X HC-HAGn+TGF-β1

Results

Under the resting state, HA and both HC-HA (PBS) and HC-HA (Gn) activatetranscript expression of BMP6 by 7- and 4-fold, respectively (FIG. 46 ).In the presence of TGFβ1, HA and both HC-HA (PBS) and HC-HA (Gn)activate transcript expression of BMP4 by 6-, 11- and 6-fold, and mRNAexpression of BMP6 by 30-, 37- and 46-fold respectively in HCF,indicating that HA and both soluble and insoluble HC-HA can upregulateBMP4/6 expression, while additional TGFβ1 further dramaticallyupregulated BMP4 and BMP6 signaling. BMP7 and BMP9 were not detected.

While TGFβ1 itself did not activated transcript expression of BMPR1A,both HC-HA (PBS) and HC-HA (Gn), but not HA activate transcriptexpression of BMPR1A by 7- and 3-fold respectively in the presence ofTGFβ1, indicating that BMPR1A may play a major role in HC-HA+TGFβ1activated BMP signaling (FIG. 47 ). In addition, TGFβ1 activates BMPR1Bby 3-fold, and BMPR2 by 3-fold, but by 4-fold on plastic with or withoutHA and both HC-HA (PBS) and HC-HA (Gn), indicating that TGFβ1 itselfnon-specifically activates mRNA expression of BMPR1B and BMPR2. HC-HA(PBS) or HC-HA (Gn) enhances transcript expression of BMPR2 to 4-fold.BMP-BMPR1A is expected to activate SMAD1/5/8 signaling while BMP-BMPRIIactivates non-SMAD signaling.

Immunofluorescence results indicate that TGFβ1 itself moderatelyactivates nuclear translocation of pSMAD1/5/8 in HCF despite thesubstrate used (FIG. 48 ). HC-HA (PBS and Gn) strongly facilitatesactivation of BMP4/6 signaling via nuclear translocation of pSMAD1/5/8,as evidenced by more nuclei having pSMAD1/5/8 and a much strongernuclear staining of pSMAD1/5/8.

ID1 is a helix-loop-helix (HLH) protein that can form heterodimers withmembers of the basic HLH family of transcription factors, a knowndownstream gene regulated by SMAD1/5/8 signaling. Our resultsdemonstrated that activation of SMAD1/5/8 resulted in 4- and 8-foldupregulation of ID1 mRNA when HCF were cultured on HC-HA (PBS) and HC-HA(Gn) respectively, indicating that SMAD1/5/8 signaling in HCF is indeedactivated by HC-HA + TGFβ (FIG. 49 ). Since ID1 has no DNA bindingactivity and therefore can inhibit the DNA binding and transcriptionalactivation ability of basic HLH proteins with which it interacts, weexpect that ID1 plays an important role in cell growth, senescence, anddifferentiation.

Example 35: Effects of HC-HA Complexes on Myofibroblast Differentiationand Reversion Of Human Corneal Fibroblasts to Keratocytes or YoungerProgenitors

Keratocytes, a unique population of neural crest-derived cells embeddedin the corneal stroma, express keratan sulfate-containing proteoglycansincluding cornea-specific keratocan. Keratocan (Kera) is acornea-specific keratan sulfate proteoglycan (KSPG) in the adultvertebrate eye. It belongs to the small leucine-rich proteoglycan (SLRP)gene family and is one of the major components of extracellular KSPG inthe vertebrate corneal stroma. Corneal KSPGs play a pivotal role inmatrix assembly, which is accountable for corneal transparency. Lumicanconstitutes about half of corneal KSPG. Most of the remaining cornealkeratan sulfate modifies keratocan. In adult tissues, keratocan islimited to corneal stroma, and keratocan expression is considered aphenotypic marker for keratocytes (Liu et al.(2003) J. Biol Chem.278(24):21672-7; Carlson et al. (2005) J Biol Chem. 280(27):25541-7). Onplastic dishes, human, bovine and rabbit keratocytes lose theircharacteristic dendritic morphology and keratocan expression whencultured in serum-containing media (Espana et al. (2003) InvestOphthalmol Vis Sci. 44 (12): 5136-41 ; Espana et al. (2004) InvestOphthalmol Vis Sci.45(9):2985-91). These exposed cells downregulate theexpression of keratan sulfate-containing proteoglycans, keratocan andCD34 and upregulate that of chondroitin-dermatan sulfate-containingproteoglycans and α-SMA, indicating that those cells become moredifferentiated.

Previous studies have shown that human (Espana et al. (2003) InvestOphthalmol Vis Sci. 44 (12): 5136-41 ; Espana et al. (2004) InvestOphthalmol Vis Sci.45(9):2985-91) and murine (Kawakita et al. (2005)JBiol Chem. 280(29):27085-92) keratocyte can maintain their phenotypewithout differentiation into α-SMA-expressing myofibroblasts whencultured on the AM stromal surface even when TGF-β is added in aserum-containing medium due to downregulation of the Smad signalingpathway. The amniotic membrane stroma can maintain keratocan expressingin cultures and prevent keratocytes from differentiating intomyofibroblasts (Kawakita et al. (2005) J Biol Chem. 280(29):27085-92) .The keratocyte maintained a dendritic morphology, continued to expresscorneal stroma-specific keratocan for at least 5 passages (at 1:2split), and did not express α-SMA under serum containing conditions oraddition of TGF-β1(Espana et al. (2004) Invest Ophthalmol VisSci.45(9):2985-91). Murine keratocytes can also be expanded on AM for atleast 8 passages without losing their normal phenotype and thatsuppression of Smad-mediated TGF-β signaling pathway is pivotal inmaintaining keratocan-expressing phenotype (Kawakita et al. (2005) JBiolChem. 280(29):27085-92). In this example, it was examined whetherimmobilized HC-HA can do the same, and if so, whether additional TGFβ1can affect their outcome.

Results

HA upregulated Keratocan mRNA expression by 4-fold (FIG. 50 ). Humancorneal fibroblasts were seeded on plastics with or without immobilizedHA for 48 h, starved without serum for 24 h, and then treated with orwithout TGFβ1 for 24 h before being harvested for mRNA quantitation anddetermination of SMAD⅔ signaling. For determination of protein of TGFβreceptors, the cells were treated with or without TGF-β1 for 48 h beforecollection of protein samples because the protein expression lags behindof mRNA expression. For TGF-β1 ELISA, the cells were treated with orwithout TGF-β1 for 24 h, and then cultured in the fesh medium for 24(and 48) h. The supernatants were collected for TGF -β1 ELISA. ForTGF-β2 and TGF-β3 ELISA, the cells were treated with or without TGF-β1for 48 h. The supernatants were collected for TGF-β2 and TGF-β3 ELISA.As expected, immobilized HC-HA promoted mRNA expression of Keratocan by14- and 16-fold, indicating those HCF are much younger when they werecultured on HC-HA without TGFβ1. After TGFβ1 challenge, the mRNAexpression of Keratocan was downregulated dramatically on plastic andHA. However, keratocan expression was still maintained at 3-fold onHC-HA(Soluble, PBS). Expression of keratocan was absent onHC-HA(Insoluble, Gn). We expect that the resultant phenotype on HC-HA(I) should be even more younger than keratocytes.

Correspondingly, immobilized HC-HA(I/S) promoted protein levels ofKeratocan by 8-and 10-fold, indicating those HCF are indeed reverted tokeratocytes when they were cultured on HC-HA(S/I) (FIG. 51 ). We did notsee any corresponding keratocan protein expression by other treatmentstested, including HA (4-fold increase of keratocan mRNA) and 4X HC-HA(PBS) (3-fold increase of keratocan mRNA), indicating such a moderateincrease of keratocan mRNA was not enough to promote correspondingprotein expression of keratocan.

Example 36: Effects of HC-HA Complexes on ESC Marker Expression in HCF

Example 35 showed a strong evidence that HCF was not only prevented fromundergoing myofibroblast differentiation under the challenge byexogenous TGF-β1 but also reverted back to keratocytes with expressionof keratocan with or without exogenous TGF- β 1. We thus examinedwhether HCF could be further reprogrammed into younger progenitors,especially when seeded on immobilized HC-HA(insoluble, GnHCl) withexogenous TGF-β1, which has been shown to suppress TGF- β signaling,promote BMP signaling, but turning off keratocan expression. We examinethe expression of a number of markers found in ESC and endothelialprogenitors and pericytes, which as we have recently reported to befound in angiogenesis progenitors. To further look into the potentialreprogramming of HCF under these conditions modulated by HC-HA, we alsoexamined expression of the four key transcription factors, i.e., Sox2,Oct4, c-Myc, and KLF4, which have been reported to play a key role ofreprogramming adult differentiated fibroblasts into iPSCs.

Results

Examination of gene expression was performed on the HCF culturesdescribed above in Example 35. The results indicated that HCF expressedmore (2- to 6-fold) ESC markers such as cMYC, KLF-4, Nanog, nestin,Oct4, Rex-1, SOX-2 and SSEA-4 on 4X HC-HA, and 2- to 4-fold more ESCmarkers even when HCF were challenged by exogenous TGF-β1when comparedto the plastic control (p<0.05, n=3) (FIG. 52 ). These results suggestthat HC-HA, especially HC-HA (insoluble), can reprogram HCF into youngerprogenitors.

Example 37: HC-HA In Solution Inhibits Osteogenesis by AffectingMC3T3-E1 Cell Viability and Differentiation

In this example, we assessed the effect of HC-HA (soluble fraction) andHA in solution on the viability and differentiation of undifferentiatedMC3T3-E1 cells. MC3T3-E1 is an osteoblastic cell line established fromC57BL/6 mouse. MC3T3-E1 cells have the capacity to differentiate intoosteoblasts and osteocytes and have been demonstrated to form calcifiedbone tissue in vitro.

MC3T3-E1 cells were cultured in the complete medium (α-MEM, 10% FBS, 100units/ml Penicillin, and 100 µg/ml streptomycin) with variousconcentrations of HA (1, 5, 25, 100 µg/ml) or HC-HA (1, 5, 25 µg/ml),with PBS as a vehicle control, and seeded in plastic cell culturetreated 96 wells at 1.6 × 10⁴ cells/ml. Cell viability was measured byMTT assays. The result showed that absorbance at 550 nm increased from24 to 48 h for all conditions except for 25 µg/ml HC-HA, suggesting thatcell proliferation proceeds normally in the control, HA from 1 to 100µg/ml, and HC-HA from 1 to 5 µg/ml (FIG. 53 ).

Next, the effect of HC-HA or HA on MC3T3-E1 differentiation intoosteoblasts was examined. MC3T3-E1 cells were re-suspended innon-induction medium (1.6 × 10⁵/ml) and seeded in 96 wells and incubateduntil confluence. The non-induction medium was removed and inductionmedium 1 (complete medium with 0.2 mM ascorbic acid 2-phosphate and 10mM glycerol 2-phospahte, manufacturer’s instruction for in vitroosteogenesis assay kit, cat# ECM810, Millipore) was added. After 12 daysof differentiation, Alizarin Red staining was used to measure andquantify osteoblast mineralization. The result showed that AR was indeedpromoted by the induction medium in the control. Consistent with a priorreport (Kawano (2011) Biochemical and Biophysical ResearchCommunications. 405: 575-580 ), 100 µg/ml HA but not 25 µg/ml HA furtherpromoted AR staining when compared to the control. In contrast, ARstaining was not reduced by 5 µg/ml HC-HA (p=0.11), but significantlyreduced by 25 µg/ml HC-HA (p=0.00002) (FIG. 54 ). These resultssuggested that increasing HC-HA concentrations during induction alsoreduced bone formation.

Example 38: Dose Dependent Response for HC-HA and AMP on OsteoblastDifferentiation Using MC3T3-E1 Model System

Previous findings showed that HC-HA and AMP dose-dependently inhibitosteoclast differentiation from RAW264.7 cells induced by RANKL (seeInternational PCT Publication No. WO 2012/149486). AMP (AmnioticMembrane Powder) is a lyophilized and then pulverized form of theamniotic membrane. In this example, IC50 of HC-HA and AMP forosteogenesis was determined and compares to that for osteoclastogenesis.

Alkaline phosphatase (ALP) assay and Alizarin Red Staining (AR-S) aretwo assays used to measure differentiation of MC3T3-E1 cells. ALP isexcreted by osteoblasts and has long been a widely recognizedbiochemical marker for osteoblast activity (Sabokbar (1994) Bone Miner.27(1):57-67), and hence serves as an early marker for osteogenesis.Alizarin Red (AR) dye forms a chelate with calcium and hence AR-S isused to visualize mineralization. Because ARS dye can be easilyextracted, it can also be converted into quantitation of mineralization(Gregory et al. (2005) Analytical Biochemistry 329: 77-84).

Part A Experimental Design MC3T3-E1 Culture

The model system of MC3T3-E1 cells from Millipore In Vitro OsteogenesisKit which consists of the base medium of α-MEM (Invitrogen, Cat. #ICM810) containing 10% FBS, 100 units/ml Penicillin, and 100 µg/mlstreptomycin, was employed. Cells were seeded at 50,000 cells/cm² andcultured in 95% air and 5% CO₂ at 37.0° C. in a 100 mL cell culture dishand passaged before confluence. Once sufficient cell numbers wereobtained then cells were seeded at 1.6×10⁵ cells/ml in 96-well culturedish with 100 µL volume of the base medium per well (52 wells). Eachconcentration was done in 4 wells with 2 wells for ALP assay and 2 wellsfor AR-S staining. Cells were cultured at 37° C. in 5% CO₂ humidifiedair and the medium was changed every 48-72 hrs until confluence.

The dosing range to be investigated was derived from the preliminarydata performed in osteoblast differentiation (see above) as well as thedose-response curves for HC-HA and AMP against osteoclastdifferentiation. Because HC-HA at 25 µg/ml significantly inhibitedMC3T3-E1 cell proliferation and differentiation into osteoblasts andcompletely inhibited osteoclast differentiation from RAW264.7 cells (seeInternational PCT Publication No. WO 2012/149486), it was selected asthe highest concentration. Because 5 µg/ml HC-HA showed less than 50%inhibition, suggesting that the IC50 of HC-HA for osteoblastdifferentiation might be higher than the IC50 in P-214, HC-HAconcentrations in the range of 0.1, 0.5, 1, 5, 10, and 25 µg/ml wereselected. Based on preliminary data on HC-HA, AMP at the followingconcentrations: 1, 5, 25, 125, 250 µg/ml was selected. Because the ALPactivity peaks at Day 12 of differentiation in MC3T3-E1 cells (Maeda(2004)Journal of Cellular Biochemistry 92:458-471;Wang, (2008) J DentRes. 87(7):650-654), we chose to study osteogenesis at Day 12 afterinduction.

Upon confluence, the medium from each well was replaced with 100 µL ofOsteogenesis Induction Medium #1. Osteogenesis Induction Medium contains0.2 mM ascorbic acid 2-phosphate and10 mM β-glycerolphosphate (in vitroosteogenesis assay kit, cat# ECM810, Millipore). 10 µL of workingsolutions of AMP and HC-HA were added into the Induction Medium #1.(Stock solutions of AMP (AMP-4; Lot #CB102971, see International PCTPublication No. WO 2012/149486) (5 mg/ml) and HC-HA (He et al. (2009) J.Biol. Chem. 284(30): 20136-20146) (250 µg/ml) in PBS were made anddiluted accordingly with appropriate culture medium (OsteogenesisInduction Medium #1) for each experimental concentration (0.1, 0.5, 1,5, 10, and 25 µg/ml for HC-HA and 1, 5, 25, 125, 250 µg/ml for AMP)).The medium was changed every 3 days.

On Differentiation Day 6, replace medium with 100 µL fresh OsteogenesisInduction Medium #2 containing ascorbic acid, β-glycerolphosphate, andmelatonin. 10 µL of working solutions of AMP and HC-HA were added intothe 100 µL the Osteogenesis Induction Medium #2(0.2 mM ascorbic acid2-phosphate, 10 mM glycerol 2-phosphate and 50 nM Melatonin Solution,manufacturer’s instruction for in vitro osteogenesis assay kit, cat#ECM810, Millipore) to make the final experimental concentrations inculture wells. The medium was changed every 2-3 days. Samples were thenassayed with ALP assay (H-156) and ARS Staining Assay following themanufacturer’s instructions (In Vitro Osteogenesis Assay Kit, (Catalog#ECM810)).

Alizarin Red S Staining

The culture medium from each well was aspirated without disturbing thecells. The cells were washed 1X with 200 µL PBS. The cells were fixed byadding 100 µL 70% ethanol and incubating at R.T. for 15 min. Fixativewas then removed and the cells were rinsed 3X (5 min each) with anexcess of distilled water without disturbing the cell monolayer. Waterwas removed and 100 µL/well Alizarin Red Stain Solution was added. Thewells were incubated at R.T. for 1 h. Excess dye was removed, and thecells were washed 4X with deionized water (gentle rocking for 5 min witheach wash). 0.1 - 0.15 mL water was added to each well to prevent cellsfrom drying. The stained cells were photographed under microscope.

Excess water was then removed from each well. 100 µL 10% acetic acid wasadded to each well and incubated with shaking for 30 min. The looselyattached monolayer was carefully removed with cell scraper and the cellsand acetic acid were transferred to a 1.5 mL microcentrifuge tube andvortexed vigorously for 30 min. The samples were heated to 85° C. for 10min, then transfer to ice for 5 min to cool the tubes. The slurry in thetubes was centrifuged at 20,000xg for 15 min. 400 µL of supernatant wasremoved and transfered to a new 1.5 mL microcentrifuge tube. The pH wasneutralized with 150 µL 10% ammonium hydroxide to within range of 4.1 -4.5. 150 µL of each sample was added to a transparent bottom 96-wellplate and read at OD405. A plot of Alizarin Red concentration vs. OD405was made.

ALP Assay (BioAssay Systems: QuantiChrom ALP Assay Kit, Cat. #:DALP-250)

Cells in each well (96-well plate) were washed with PBS and lysed in 100µL 0.2% Triton X-100 in distilled water by shaking for 20 min at R.T.200 µL distilled water and 200 µL Calibrator solution (supplied by kit)were transferred into separate wells for controls. 50 µL samples weretransferred into separate wells. 150 µL Working solution (200 µL AssayBuffer, 5 µL Mg Acetate (5 mM), 2 µL pNPP liquid substrate (10 mM)) wasadded to the sample wells (final reaction volume was 200 µL). The platewas tapped briefly to mix. OD₄₀₅ was read at 0 min and 4 min on platereader.

Results Phase Contrast Microscopy

The negative control maintained a hexagonal shape through 13 days ofinduction (FIG. 55 ). The monolayer was smoother than the positivecontrol, suggesting that more cells or more pile up of cells occurred inthe latter. Cells in the positive control became fusiform in shape afterbeginning of induction. With more time, a spindle-like ring developedalong the edge (~ 2-3 mm away) of the plastic culture around the 4^(th)day of induction.

From 0.1 µg/ml to 10 µg/ml of HC-HA, the cell monolayer did not differfrom that of the positive control, suggesting that HC-HA at theseconcentrations did not negatively affect the induction (FIG. 55A). Likethe positive control, cells also developed a fusiform shape and themonolayer developed a spindle-like ring. However, at 25 µg/ml, adramatic decrease in cell density and change in cell morphology wasobserved on D13 of induction.

At concentrations above 25 µg/ml, AMP deposited particles that settledon the monolayer (FIG. 55B). Because AMP concentration was replenishedafter each medium change, AMP deposit on top the monolayer increasedthrough induction period. Treatment with AMP below 125 µg/ml did notaffect cell morphology as cells also developed fusiform shapes withspindle rings, suggesting that AMP did not negatively influenceinduction. At concentrations above 125 µg/ml, AMP particle densityincreased to the extent that obscured visual observation of spindle ringformation. However, cell density and morphology remained unchanged fromthat of the positive controls, also confirming that AMP did notnegatively affect induction.

Alizarin Red Staining

The negative control yielded a blue-gray background color with parts ofthe monolayer showing a light pink. In contrast, the positive controlyielded a rose pink background (FIG. 56 ).

0.1 µg/ml HC-HA yielded a rust red color with the visible spindle ringstaining red-brown, which was dramatically different from that of thepositive control (FIG. 56A). This trend continued from 0.5 µg/ml to 1µg/ml with a slight lightening of color at 10 µg/ml, suggesting that themineralization was maintained from 0.1 to 5 ug/ml and that there mightbe a dose-dependent relationship between 0 and 0.1 ug/ml. At 25 µg/mlHC-HA, the rust red background disappeared and returned to a lightpurple-pink with noticeable white gaps in between the cell junction.

AMP at 1 µg/ml to 125 µg/ml, yielded a dose dependent change of colorfrom a lighter rust brown (1 µg/ml), which appeared to be lighter than0.1 ug/ml HC-HA, suggesting that the dosing response was more gradual,to a rust red background (5 µg/ml & 25 µg/ml), red-brown (125 µg/ml),and dramatically increased to dark red-brown at 250 µg/ml (FIG. 56B). Itis noted that AMP particles found on the top of cell monolayers treatedwith more than 5 µg/ml of AMP, and the particle size was smaller thanthe cell itself, and appeared to match the color of the stainedbackground.

By quantitative analysis of OD value, ARS staining with 0.1 µg/ml HC-HAtreatment increased 3x from positive control with statisticalsignificance (p < 0.05) (FIG. 57A). Some variation in concentrations 0.1µg/ml to 10 µg/ml was observed, which may be attributed to small samplesize (N=2). A dramatic decrease OD value at 25 µg/ml HC-HA treatment wasobserved. For AMP, treatment with 125 µg/ml of AMP more than doubled theamount of mineralization from positive control and was statisticallysignificant (p < 0.05) (FIG. 57B). A small decrease in OD value from thepositive control was seen in 1 µg/ml AMP, and from 1 µg/ml to 25 µg/ml,there was a small dose dependent increase in OD values. OD₄₀₅ decreasedin 250 µg/ml AMP compared to 125 µg/ml. Some variation in may beattributed to small sample size (N=2).

ALP Staining

In both AM derivatives group, the negative control showed 5-fold moreALP activity than the positive control, which could have occurred fromloss of sample in the negative control, which decreased the sample size(FIG. 58 ). The smaller sample size contributed to the standarddeviation value being 8x higher for the negative control than thepositive control, and this much larger variation could contribute to theincrease.

Treatment of induced MC3T3-E1 with any amount of HC-HA decreased ALPactivity compared to the positive and negative control (FIG. 58A). ALPactivity varied between the different concentration groups until 25µg/ml where ALP activity significantly dropped. It is worthwhile to notethat there was very little variation from the mean in each of theexperimental groups and the positive control.

At 1 µg/ml, ALP activity was almost 4-fold higher than the positivecontrol (FIG. 58B). This phenomenon was blocked at 5 µg/ml and 25 µg/mlwith ALP activity decreased almost 3x from the positive control.Increasing the concentration to 125 µg/ml to 250 µg/ml restored ALPactivity to levels close to 1 µg/ml. It thus appeared that ALP activitywas not congruent with the amount of mineralization.

Results Summary

Compared to the negative control, the positive control exhibited morefusiform cells and formed “ring” around the edge of the plastic well(FIG. 55A), a change of color by alizarin staining (FIG. 56A), and adetectable but not significant change of OD₄₀₅ (FIG. 57A). Previously,MC3T3-E1 cells seeded at 5x10⁴ cells/35 mm plastic dish also revealedformation of layered collagen fibrils after day 4, layered fibrils byday 18, and formation of nodular regions by day 21 of induction. (Sudo(1983) J. Cell. Biol. 96: 191-198). This prior study did not note thesame ring formation as we observed. [Alternatively, they may interpretthe ring as layered collagen fibril. If this were the case, the ringarea should be prone for mineralization.]

Alizarin red staining has been described as a crimson red color in theliterature to signify mineralization. Mineralization and osteoblasticnodules were described to be stained a deep red and the intensity ofcolor increased with mineralization (Wang, (2006) Biotechnol. Prog.22(6):1697-701; Zhao, 2007). ARS staining is read at 405 nm, whichcorresponds to a violet color in the visible spectrum. Unlike ourresults, color photographs of ARS staining of MC3T3-E1 mineralizationfrom published data did not show a rust-red or red-brown color in themonolayer. The darker color in our results indicates more ARS stainingand thus more osteogenesis compared with published results. The amountof OD change in ARS staining and quantitation varied depending on theculture conditions and cell type. Human mesenchymal stem cells (hMSCs)cultured in 6-well (10 cm²/well) for 30 days attained an increase fromOD₄₀₅ of 0.5 to 4 (Gregory et al. (2005) Analytical Biochemistry 329:77-84). MC3T3-E1 cells cultured for 28 days (α-MEM, ascorbic acid,βglycerolphosphate) in 24-well plates attained an OD of 0.6. However,Day 16 and Day 26 OD were much lower than Day 28 at below 0.05 and 0.2,respectively (Burkhardt (2006) University of Basel, Master Thesis). Thelack of dramatic color change in the positive control might be due tothe time of D13, which was too short for ARS although it is optimal forALP.

0.1 µg/ml HC-HA also induced “ring change” (FIG. 55A), a clear increaseof color (FIG. 56A), and 3X higher OD value than the positive control (p< 0.05) (FIG. 57A), suggesting that HC-HA at the lower dose promotesmineralization and that a dose-response curve exists between 0 and 0.1ug/ml.

HC-HA from 0.5 to 10 µg/ml also showed “rings” (FIG. 55A), maintainedthe same color as 0.1 ug/ml (FIG. 56A), and yielded OD₄₀₅ without astatistical significance. HC-HA at 25 µg/ml decreased cell density,changed the cell morphology, lost “ring” (FIG. 55A), did not yield anycolor change (resembling the negative control) (FIG. 56A), and generatednegligible OD₄₀₅ (like the negative control).

AMP from 25 µg/ml left particles (FIG. 55B), from 1 µg/ml increased thecolor with a positive dose-responsive relationship (FIG. 56B), but OD₄₀₅showed an increase that was not statistically significant until 125µg/ml AMP (p < 0.05), and then declined at 250 µg/ml, which was notconsistent with the color change. Unlike HC-HA, AMP at the higher dosedid not cause any ill effects on cell morphology.

Part B

The Alizarin Red Staining method was then improved by increasing thesample size and incorporating the methodology with Gregory et al.((2005) Analytical Biochemistry 329: 77-84) used for Alizarin stainingof human MSC and other methods known in the art. A comparison of themethod of Gregory et al, our previous method described above, and thenew method outlined in this example is provided in the table below.Previous studies showed that MC3T3-E1 differentiation under inductioncan be subdivided into three stages, i.e., proliferation (day 1 to 9),ECM formation (day 9 to day 16), and mineralization (deposit minerals informed ECM) (day 16+). (Quarles et al. (1992) Bone Miner Res.7(6):683-92; Hong et al. (2010) Exp Cell Res. 316(14):2291-300). Tocompare studies from different groups, it was thus important to time theevent starting from confluence as Day 0.

TABLE 5 Gregory et al. 2005 Previous Method New Extraction Method CellType Human MSC MC3T3-E1 MC3T3-E1 Culturing conditions 5000 cells per cm²(6-well) αMEM, 20% FCS, streptomycin, penicillin, glutamine; induction:sodium glycerolphosphate, ascorbic acid, dexamethasone 3.1×10⁴ cells/cm²(96-well) αMEM, 10% FBS, streptomycin, penicillin, glutamine; induction:βglycerophosphate, ascorbic acid, melatonin 3.1×10⁴ cells/cm² (96-well)αMEM, 10% FBS, streptomycin, penicillin, glutamine; induction:βglycerophosphate, ascorbic acid, melatonin ARS Time Day 0 - Day 30 Day18 Day 18 ARS Staining & Quant. Steps 1. Wash with PBS ← Same ←Same 2.Fix in 10% formaldehyde RT, 15 min. Fix in 4% Paraformaldehyde RT, 15min. Fix 4% Paraformaldehyde RT, 15 min 3. Wash 2x with dH2O ← ← 4. 1mL/well 40 mM ARS RT, gentle shaking, 20 min. 100 µL/well 40 mM ARS perwell RT gentle shaking for 1 h. ← 5. Wash 4X with dH2O, shaking (5 min).Remove excess water (tip plate) ← ← 6. View by phase microscopy. ← ←7. * Add 800 µL/well 10% acetic acid RT, shaking (30 Add 300 µL/well 10%acetic acid RT, shaking (30 min) Incubate samples with 350 µL/well 4 Mguanidine HCl @ min) 37° C. O/N 8. Scrape monolayers w/ cell scraperTransfer ➔ 1.5-mL tube ← Transfer 150 µL solution in duplicate to read9. Vortex for 30 s ← 10. Overlay the slurry with 500 µL mineral oil,heat to 85° C. for 10 min Heat to 85° C. for 10 min (with the cap on).11. Transfer to ice ➔ open when cooled. ← 12. Centrifuged at 20,000 gfor 15 min ← 13. * Remove 500 µL of 800 µL supernatant to a new 1.5-mLtube. Remove 255 µL of 300 uL supernatant to a new 1.5-mL tube. 14.Neutralize pH with 200 µL of 10% ammonium hydroxide (pH 4.1 -4.5)Neutralize pH with 95 µL 10% ammonium hydroxide (pH 4.1 - 4.5) 15. * Add150 µL in triplicates in opaque-walled, transparent-bottomed 96-wellplates. Read at 405 nm. Add 150 µL of standard/sample in doubles toopaque-walled, transparent bottom 96-well plate. Read at 405 nm. Read at490 nm in opaque-walled, transparent bottom 96-well plate. *Steps withdifferences between the 3 methods for ARS staining and quantitation.

Experimental Design

Cells were cultured and stimulated in differentiation medium asdescribed in Part A and Table 5. Differentiation medium was changedevery 3 days for 18 days. 0.1 µg/ml HC-HA and 125 µg/ml AMP wereemployed for the assay. The ARS assay was performed as described in PartA with the changes as noted in Table 5.

Results Cell Morphology and Ring Formation

Uninduced cells attained a flat cuboidal shape after seeding (FIG. 59A).Cell border became more defined on Day 4 with raised edges, and somecells developed fusiform shapes on Day 6. No spindle cells or multiplelayers developed.

From Day 1 to Day 3, cells maintained a cuboid shape and monolayerremained flat(FIG. 59B). By day 3, cell borders became more distinct andcell edges became raised. In addition, small round cell-like structureswere visible on the monolayer (indicated by black circle). Cellmorphology changed by day 4 with the appearance of fusiform shaped cellsand cells organized in multiple layers. The appearance of the smallround cell-like structures continued to increase through Day 6 and Day7.

Cell morphology change in induced cells treated with HC-HA mirrored thepositive control’s changes (FIG. 60A). HC-HA did not leave particles onthe monolayer like AMP. Like the positive control, small round cell-likestructures (shown in black circle) appeared on Day 3 and continued toincrease to Day 7.

AMP particles (noted with black arrow) settled on top of the monolayerand obstructed observation of the monolayer underneath (FIG. 60B). Areasdevoid of AMP particles on Day 0 and Day 1 showed round and cuboidalshapes. On Day 2, some fusiform shaped cells appeared on the monolayer.It was difficult to identify and distinguish the small round cell-likestructures from smaller AMP particles and the development of thesestructures remains unknown. On Day 5, fusiform shapes elongated to formspindle-like cells. On Day 6, long spindle cells formed web-likeinteractions with AMP particles (shown in black circle).

On Day 3, fusiform shaped cells developed near the edge of the well (1 -2 mm from the edge) (FIG. 61A). Spindle cells and ring formation did notdevelop from Day 4 onwards. Cells appeared to overlap each other at theedge and grow into fusiform shapes. From Day 0 to Day 2, there are nospindle-like cells along the well edge (FIG. 61B). On Day 3, similarfusiform cells piled up in a ring configuration were noted in the edge.From Day 5, these cells were concentrated as a prominent ring about 2 mmfrom the edge. On Day 6, monolayers show detachment from the plasticsurface along certain areas near the edge (indicated by white ➔).

Cells remained smooth and cuboidal along the edge until Day 2 (FIG.62A). Fusiform shaped cells developed near the edge on Day 2. Some smallround cell-like structures were also visible near the edge at the time.Spindle-like cells developed on Day 3 and continued to thicken in a ringaround the well edge to Day 5. Monolayer detachment from the plasticwell was observed on Day 6 in areas near the edge (noted with whitearrow). Fusiform shaped cells appeared near the well edge on Day 2 (FIG.62B). Spindle-like cells developed from the edge on Day 3 (about 1 to 2mm away) and a ring of spindle-like cells formed by Day 5. Monolayerdetachment from the plastic well was observed on Day 5 in some areasnear the edge. Detachment continued on Day 6, but the monolayers did notdetach as much as the HC-HA treated cells and the positive controlcells.

ARS Staining and Quantitation

The negative controls monolayer stained a light pink color in some areas(FIG. 63 ). The positive control stained a light pink in the center butshowed a bright crimson red color in the spindle ring area, indicatingMC3T3-E1 cells deposit mineralization heavily in ring rather than therest of the monolayer. Both intensity and color of staining in thecenter monolayer and the spindle ring between 0.1 µg/ml HC-HA andpositive control was the same. AMP particles on top of the cells staineda red-brown color. Visual observation of cells underneath was obstructedby the stained particles, but openings showed lack of prominent cellmonolayer with some sparse cells staining a light pink color similar tothe negative control. Since AMP treated cells did not show a visiblespindle ring, and the ARS did not stain a crimson red around the edgesimilar to the positive control.

GnHCl treatment solubilized the cell matrix and removed crimson red ARSdye while leaving the monolayer intact in both the positive control andHC-HA treated cells. GnHCl digested and denatured the cell protein,leaving the extracellular matrix behind. In the AMP experimental group,AMP particle density decreased but most particles still remained on thebottom of the well. With the long culture time, AMP particles may formtight interactions with the ECM matrix that was not dissolved by theGnHCl. The particles that once stained a bright red-brown now showed alight brown like the color AMP naturally exhibits. A distinct monolayerstructure, however, was not observed; this supported the observation ofa monolayer of cells in the gaps between the AMP. The cells may havemigrated from the monolayer into the AMP particles and used it as ascaffold for differentiation and mineralization.

ARS standard showed a progression from crimson red to a cream pink colorthrough serial dilution from 2 mM to 31.3 µM (FIG. 64A). There was anoticeable color change between HC-HA and AMP treated samples. HC-HAtreated cell extracts showed a clear cream color while AMP treated cellextracts showed a light cream pink color. The positive control alsoshowed the same color and level of intensity of color as the HC-HAtreated cells, while the negative control showed lighter color andresembled the blank (not shown). The OD₄₀₅ values stayed in the samerange as the values from in the previous example (FIG. 64B). Compared tothe negative control, the positive control showed a staticallysignificant 2-fold decrease in OD. HC-HA-treated (0.1 µg/ml) cellsshowed slight increase in OD than the positive control, but thedifference is not statistically significant. AMP (125 µg/ml) treatmentslightly decreased OD values when compared to the positive control, butthis decrease was not statistically significant.

ARS standard showed a progression from crimson red to a cream pink colorthrough serial dilution from 2 mM to 31.3 µM (FIG. 65A). The negativecontrol extract showed a light cream color slightly darker than theblank (not shown). The positive control showed a light golden color andthe color was visibly darker than the negative control. HC-HA alsoshowed a light golden color at the same intensity as the positivecontrol. AMP treated extracts showed an orange-golden color that wasdarker than both the positive and HC-HA extract groups. OD₄₀₅ valuesstayed in the same range with the highest at around 0.25 (FIG. 65B).Negative controls showed a negligible OD value close to 0. The positivecontrols and HC-HA (0.1 µg/ml) treated extracts showed an average ODvalue close to 0.05. AMP-treated (125 µg/ml) extracts showed astatically significant 5-fold increase in OD (P = 0.039) from both thepositive and HC-HA treated groups.

Summary Cell Morphology

MC3T3-E1 cells cultured in αMEM w/ 10% FBS grew to confluence anddeveloped cuboidal shape. Like findings in Aim #1 and #2, cells did notdifferentiate without the addition of ascorbic acid,β-glycerolphosphate, and melatonin. Without induction medium, spindlecells and spindle rings did not form (FIG. 59 ). MC3T3-E1 cells wereinduced into differentiation with ascorbic acid, β-glycerolphosphate andmelatonin. After seeding, a smooth monolayer formed with cuboidal shapedcells. After 3 days of induction, cells attained a fusiform shape. Byday 5, cells elongated and became spindle-like.

In this example, spindle rings developed on Day 3 of induction, withspindle like cells forming 1 to 2 mm from the well wall. On Day 6,detachment of the monolayer from the well edge and plastic bottom wasobserved (FIG. 60 ). Small round cell-like structures developed on themonolayer beginning on day 2 and increased in number to day 6 (FIG. 59). They did not float in the culture medium and were firmly attached inthe monolayer, resting mostly in between the cell borders. Thesestructures may represent matrix vesicles (MVs) are extracellular,membrane-invested particles located at sites of initial calcification incartilage and bone. Matrix vesicle synthesis occurs through budding andpinching-off of vesicles from specific regions of the outer plasmamembranes of differentiating growth plate chondrocytes and osteoblasts(Anderson et al. (2003) Curr Rheumatol Rep. 5(3):222-6).

Treatment with HC-HA did not alter MC3T3-E1 cell morphological changethrough differentiation (FIG. 60 ). Formation of fusiform shaped cells,spindle-like cells and spindle ring (FIG. 61 ) followed the reportedtime course of the positive control. AMP particles settled on top of thecell monolayer similar to pervious findings (FIG. 60 ). This impededfull observation of the cell monolayer and cell morphology changethrough induction. However, some observations were noted throughopenings where AMP particles did not settle. Unlike HC-HA, AMP treatmentsped up cell morphological change and some fusiform cells were visible aday earlier on induction Day 2. AMP treated cells formed a spindle ringsimilar to HC-HA treated cells and the positive control (FIG. 62 ).However, the monolayer detached earlier (FIG. 62 ) then the other twoexperimental groups (on Day 5 instead of Day 6).

ARS Staining and Quantitation

ARS staining showed drastically different color than previous reported(FIG. 66 ). ARS stained a light pink instead of blue-grey color in thenegative control cell monolayers. ARS staining also showed a brightcrimson red concentrated in the spindle ring instead of a rust-browncolor from previous results in the positive controls and HC-HA treatedcells. AMP particles stained a red-brown instead of a dark brown, andthe cells underneath was stained light pink instead of a rust-brown.Degradation of ARS Solution may have contributed to the color change.

After Acetic Acid extraction, the monolayer still appeared to havesignificant amount of stained color left. Acetic Acid is not effectivein completely removing ARS stain from the monolayer. With Acetic Acidextraction, there was a statistically significant difference between theOD values of the positive and negative control (FIG. 68 ) that matchedthe visual observation of more ARS staining in the positive control(FIG. 66 ). However, acetic acid extraction was not effective in showinga statistically significant increase in AMP’s OD compared to thepositive and HC-HA groups (FIG. 66 ) despite visual observation of AMPgroups having more color in the assay extracts than the other two groups(FIG. 67 ). AMP particle deposits may have made it more difficult toremove ARS from the AMP groups. Mineralized matrix and cells could alsomay have interactions with the AMP particles that hinder acetic acidextraction of ARS.

GnHCl removed ARS from cell monolayer more completely than Acetic Acidtreatment (FIG. 69 ). Additionally, color was solubilized in GnHClsolution without the use of a cell scraper to remove the monolayer. Bothmethods seem to have left particles invisible to the naked eye andunaffected by centrifuge. For GnHCl, this could be calcium and dissolvedmatrix that forms fine particles. This caused variations between theduplicates in each sample. Reading at 670 nm to remove the particlessolved this problem for the GnHCl extraction method.

GnHCl extraction could not establish a quantifiable statisticalsignificance between the negative and positive control (FIG. 71 ) tomatch the visual observation of the ARS staining. GnHCl extraction alsoshowed that 0.1 µg/ml HC-HA did not promote more mineralization indifferentiating MC3T3-E1 cells (FIG. 71 ), and this matched the resultsof the Acetic Acid extraction. From looking at the assay extract colorsin 96-wells, the positive control and HC-HA wells showed no differencein color or intensity (FIG. 70 ).

GnHCl extraction showed that 125 µg/ml AMP promoted more mineralizationin differentiating MC3T3-E1 cells than the positive control (FIG. 71 ).This matched both the ARS staining of the monolayer (FIG. 66 ) and thevisual observation of the extracts in 96-well assay plates (FIG. 70 ),where AMP showed a much deeper golden-yellow color than the HC-HA orpositive control.

From these results, GnHCl is the better extraction method because itremoves ARS stain more completely from the monolayer and leaving itintact; there is less technical error because the monolayer does notneed to be scraped from the well. The extract color can be quantifiedthrough spectrophotometer reading.

Part C

The preliminary study above did not exhibit statistically significantresults on the dose-response curves for HC-HA and AMP on osteoblastdifferentiation due to the small sample size and incomplete developmentof the ARS assay. This suggested that 0.1 µg/ml HC-HA may enhancemineralization. Furthermore, HC-HA at 10 µg/ml to 25 µg/ml might affectcell viability and reduce mineralization. The dose curve for HC-HAshould include a lower concentration below 0.1 µg/ml as well asconcentrations above 10 µg/ml. Unlike HC-HA, AMP at 5 µg/ml to 125 µg/mlmay promote mineralization. In this experiment, the dose response ofHC-HA and AMP was retested using the method of Part B. In thisexperiment, ARS stain was used to assay at Day 15, and the revisedprotocol using 10% acetic acid to stain and quantitate was employed.

Experimental Design

The same model system as shown in Part B based on 3T3-E1 cells was usedby seeding 3×10⁴ cells/cm²/well in 96 well plates in αMEM medium w/ 10%FBS. Upon confluence, cells were induced to differentiation by addingascorbic acid, β-glycerolphosphate, melatonin induction medium. For eachcondition, N=3 was tested. After confluence (Day 0 = seeding), HC-HA wasadded at 0.02 µg/ml, 0.1 µg/ml, 1 µg/ml, 5 µg/ml, and 25 µg/ml while AMPwas added at 1 µg/ml, 5 µg/ml, 25 µg/ml, and 125 µg/ml. For ARSstaining, the modified method of Part B was employed.

Results

Negative control MC3T3-E1 cells did not develop spindle-like shapes orspindle rings throughout culturing (FIG. 66A). The monlayer center andperipheral stained a beige color. The positive control cells developedfusiform and spindle-like cells, with the appearance of spindle ringsaround D5 of culturing (D4 of induction). ARS staining showed a lightmaroon color in the center and was mostly concentrated in the spindlerings with a dark crimson color. Increasing HC-HA concentration to 1.25µg/ml had no effect on cell morphology or ARS staining intensity andpattern. At 2.5 µg/ml, MC3T3-E1 cells’ spindle ring started to degradeand the ARS color changed from crimson to a red-brown color. By 20µg/ml, cells lost their fusifrom and spindle shapes; cell edges werealso less defined and raised. Cells density decreased and the monolayerdid not appear raised like before. GnHCl successfully extracted the ARSdye and the coefficient of variation in OD₄₅₀ values ranged from 5% to19%. The positive control showed statistically significant increased ODvalues (FIG. 66B).

Negative control MC3T3-E1 cells did not develop spindle-like shapes orspindle rings throughout culturing (FIG. 67A). The monlayer center andperipheral stained a beige color. The positive control cells developedfusiform and spindle-like cells, with the appearance of spindle ringsaround D5 of culturing (D4 of induction). ARS staining showed a lightmaroon color in the center of the monolayer. AMP treatment left AMPparticles that settled on top of the cell monolayer and obscuredobservation of the monolayer from concentration 62.5 µg/ml upwards.MC3T3-E1 cells treated with only AMP and no induction showed no spindlerings along the edge and ARS staining showed a dark crimson with a lightpink background. From 7.8 µg/ml to 31.25 µg/ml, the AMP particles didnot completely cover the monolayer and cells showed fusiform and spindleshapes. Along the edge, spindle rings could be seen. ARS staining showedthe center monolayer staining a light maroon and the dye concentratedalong the spindle ring to a dark crimson color like the positivecontrol. GnHCl successfully extracted the ARS dye and the coefficient ofvariation in OD₄₅₀ values ranged from 3% to 10% (FIG. 67B).

Results Summary Cell Morphology/ARS Staining

Similar to previous results, MC3T3-E1 differentiation progresses fromcells changing from cuboidal shape to fusiform and spindle shapes. Withincreasing induction time, spindle rings form along the well edge ( ~ 2mm away) and contracts the monolayer with time. Unlike the positivecontrol, the negative control never developed spindle-like cells orspindle ring. While the negative control monolayer stained a uniformbeige color, the positive control showed a light maroon/pink in thecenter and concentrated dark crimson in the spindle rings.

At 10 µg/ml HC-HA, cell morphology changed from lower concentrations,with less cells showing the fusiform and spindle shapes. Cell densitydecreased and the monolayer looked less raised. At 20 µg/ml HC-HA, celldensity decreased dramatically and few cells were spindle shaped. Themonolayer looked smooth like the negative control. For bothconcentrations, these changes were noticed starting on D5 of culturingand induction. In both concentrations, the spindle rings were eitherpoorly formed or non-existant. Increasing HC-HA concentration caused thedisintegration of the spindle ring at a concentration of 2.5 µg/ml andupwards.

In all AMP treated cells, areas around where AMP particles settledstained dark crimson. Around the edge of the well, no monolayer could beseen through the openings of AMP particles. Uninduced MC3T3-E1 treatedwith 125 µg/ml AMP showed staining similar to induced MC3T3-E1 treatedwith 125 µg/ml AMP. The cells with settled AMP stained dark crimson inpatches, with no observation of a stained monolayer underneath. From 7.8µg/ml to 15.6 µg/ml AMP, cell morphology was visible. Cells showedfusiform and spindle shapes with spindle rings formed along the edge ofthe well. ARS staining resembled the positive control with light marooncolor in the center and dark crimson in the spindle ring areas.

GnHCl treatment was successful in extracting ARS dye from the stainedmonolayer. The extraction showed a statistically significant (p < 0.01)2-fold increase in OD₄₅₀ from negative control to positive control.HC-HA treated cells showed a trend of decreasing mineralization withincreasing HC-HA concentration. At 10 µg/ml and 20 µg/ml HC-HA, therewas a statistically significant (p < 0.05) decrease in mineralizationfrom the positive control. AMP dose-dependently increased mineralizationof differentiating MC3T3-E1 cells.

Uninduced cells treated with AMP also showed mineralization, and itwould appear AMP induced and promoted mineralization more than positivecontrol (p < 0.01). Also, at 125 µg/ml AMP, treatment to uninduced cellsshowed more mineralization than cells cultured in induction medium ( p <0.05).

Example 39. Effect of AMP on Osteoblast Differentiation

Although ARS staining showed a clear dose-dependent increase of stainingby AMP, and exhibited a statistically significant increase inmineralization at 125 µg/ml, it was unclear whether such a change iscaused by non-specific binding of ARS to AMP. Because AMP acteddifferently from HC-HA especially at the high dose, i.e., promotingmineralization but not inhibiting it, it was important to rule outwhether AMP’s action depends on cell direct contact with AMP. Thisquestion was addressed by the use of transwells with a 3 µm pore size,which is sufficient for HC-HA to pass through but small enough topreclude AMP particles. Because the available transwell plate with thispore size fits in a 24-well plate, the culture conditions of the assaywere altered accordingly. A concentration of 125 µg/ml AMP was employedfor the assay.

Experimental Design

MC3T3-E1 cells (Cells at P2; ATCC, catalog number: CRL-2593™) wereseeded onto 12-well flat, transparent bottomed wells at a density of1×10⁵ cells/ml. The same AMP stock solution (AMP-4; Lot #CB102971) asused above was prepared as 5 mg/ml in PBS. For wells without Transwells,17.5 µL of AMP stock was added in 0.7 ml of the culture medium (base orinduction medium) to achieve 125 µg/ml AMP concentration in either α-MEMw/ 10% FBS (without induction) or induction medium #1 followed by #2(with induction). For wells with Transwells, 17.5 µL of AMP stock wasadded to directly into center of Transwell membrane in the same manneras described above. Culture mediums (α-MEM with 10% FBS; InductionMedium #1 and #2 as described above) were changed every 3 days after D0of induction. ARS staining and quantitation procedures were performed asdescribed above except that cell monolayers were fixed with 4%paraformaldehyde instead of 70% ethanol, and stained for 2 h instead of1 h.

Results

All induced cells developed ring formation on D4 of induction (FIG. 68). Rings were composed of layers of cells around the edge of the plasticwells, about 2-3 mm away. They grew in layers, curled up, and then themonolayer detached in many wells from the plastic. Without induction,cells maintained mostly hexagonal shapes with some fusiform shapes withincreasing culture period, and the monolayer remained smooth compared toinduced cells (FIG. 69 ). Induction rendered the cells with a fusiformshape and the monolayer became raised and borders between cells becamemore distinct. With induction, by day 4, a spindle-like ring wasobserved developing along the edge of the culture plate. Treatment withAMP did not affect cell viability; nor did it affect the ring formation,suggesting that AMP did not negatively affect induction. The addition oftranswells did not affect cell growth or morphology.

Without transwells, AMP at 125 µg/ml left particles settled on the cellmonolayer (FIG. 69A). Without induction, AMP, by itself, did not causecells to develop spindle shape and did not generate ring, thussuggesting that AMP alone was not sufficient to cause induction, similarto the negative control. With induction, 125 µg/ml of AMP did cause thecell morphology change like the positive control, suggesting that AMPitself did not negatively affect induction. Through transwells, 125µg/ml AMP left negligible particles on the cell monolayer (FIG. 69B).Without induction, AMP was not sufficient in inducing cells todifferentiate and cells resembled the negative control. With inductionand 125 µg/ml of AMP, cells developed fusiform and spindle-like shapesand by D14 resembled the positive control, again suggesting that AMP didnot negatively affect induction.

The negative control yielded a taupe background color with patches oflight pink in the monolayer, which differed from the blue-gray color ofthe negative control in the previous experiment (FIG. 70 ). Withoutinduction but with transwell, monolayers stained a taupe backgroundsimilar to the negative control even when treated with AMP, suggestingthat AMP itself did not cause induction. Without transwell, compared tothe negataive control, there were zones exhibiting a notable decrease ofbackground color, suggesting that AMP particles settled on monolayermight have blocked the color (marked by **), and AMP itself did notgenerate positive color suggesting mineralization.

The positive control yielded rose pink with the ring stained rust red,which was darker than rose-red color from the positive control in theprevious experiment. The addition of a transwell did not affect color ofthe positive control, confirming that transwell itself did not affectinduction. With AMP but without transwell, monolayers yielded a strongerrust red color than the positive control, with the ring stained darker,suggesting that AMP exerted additional positive induction. In contrast,with the transwell, the intensitiy of the background color decreased tothe level of the positive control while the ring maintaining the samecolor, suggesting that transwell exerted a negative influence on AMP’seffect on induction.

Quantitation results were not adequate in providing statisticalsignificance, and did not match visual analysis of ARS staining (FIG. 71). However, the overall trend suggested that the positive control showedmore mineralization than the negative control. Furthermore, there was atrend suggesting the less OD₄₀₅ when transwell was included in thepresence of AMP and induction.

Results Summary

The ring formation (FIG. 69A) was easy to observe presumably because thesize of dish was bigger. However, probably due to the change offixative, the background color for the negative control was differentfrom the prior experiment. Furthermore, there was a more dramatic colorchange between negative and positive controls, especially in the ringarea (FIG. 70 ). The introduction of a transwell in the positive controldid not affect cell morphology (FIG. 69B) or ARS staining color (FIG. 70).

Without induction, AMP without transwell clearly blocked the positiveARS staining (FIG. 70 ), and AMP with transwell showed the same color asthe negative control. AMP itself does not cause non-specific ARS anddoes not cause any induction. With induction, AMP without transwellcaused more color than the positive control. In contrast, AMP withtranswell seemed to yield the same color as the positive control.

Previous ARS quantitation results showed 125 µg/ml AMP promoting 3X moremineralization than positive control (p < 0.05) (FIG. 67 ), but not soin FIG. 71 .

There was no difference in cell morphology between cells directlytreated with AMP and cells treated with AMP through transwells (FIGS.69A, 69B). Both cell groups developed ring formation and visualobservation showed no perceivable difference between the ringstructures. ARS staining showed negligible difference in backgroundcolor between the two experimental groups. However, cells directlytreated with AMP showed a more diffuse ring formation, which could be ascattering effect from the AMP particles (FIG. 70 ).

AMP does not need direct contact with MC3T3-E1 cells to affectmineralization. While it was unclear from this experiment AMP promotesmineralization, AMP has been shown to not affect cell morphology or cellviability (FIG. 69 ).

Part B

Our results showed that 125 µg/mL AMP significantly increasedmineralization of MC3T3-E1 cells (FIG. 65 ) when compared to thepositive control on Day 15 of growth and differentiation. AMP wasdelivered directly in culture medium and AMP particles settled on top ofthe cell monolayer; therefore, AMP’s effect in Aim 1B necessitateddirect contact.

In Part A, we sought to investigate whether AMP’s effect is throughacting as a scaffold for the differentiating MC3T3-E1 cells or iffactors are released from the particles to promote mineralization.However, the small sample size and technical errors in ARS stainextraction with 10% acetic acid affected the data and no statisticalsignificance was found. The experiment was repeated using the improvedtechnical methods of ARS staining and extraction developed in Example 38with 4 M guanidine HCl.

Experimental Design

MC3T3-E1 cells were seeded at 3×10⁴ cells/cm²/well in 24-well with αMEMmedium plus 10% FBS as described above. Upon confluence, cells wereinduced to differentiation by adding ascorbic acid, β-glycerolphosphate,melatonin. For each condition, N=3 was tested. Day 0 counted as the dayof cell seeding, and induction followed after cell confluence. Totalinduction time = 15 Days. There were two experimental groups: AMPdirectly added to induction medium and AMP delivered through transwell.AMP concentration was kept the same as before at 125 µg/ml. A negativecontrol (without induction) was added with or without AMP but withoutinsert. ARS staining and quantitation on Day 15 of induction wereperformed as described above.

Results

Without induction medium, negative control cells maintained hexagonalshapes with some fusiform shapes (FIG. 72A). Spindle-like shapes werenot observed, and no spindle ring formed along the periphery. ARSstained the monolayer a light pink. With induction, positive controlcells attained spindle like shapes. Cell borders were more prominent andraised; a spindle ring formed along the periphery near the well edge.The monolayer center stained a maroon color, and the ARS stainconcentrated in the spindle ring with an intense crimson red color.Treatment with AMP directly caused AMP particles to settle on themonolayer and obscured the morphology of the cells. However, near theculture well edge, gaps between the AMP particles showed lack of aprominent monolayer underneath in both groups. There was no differencein cell morphology between AMP treatment alone and AMP treatment withinduction. The cells that were visible were spindle like in shape. Nospindle ring like the positive control was observed. ARS staining showeda crimson red staining in the center with reddish-brown staining alongthe periphery. Staining color and patterns were indistinguishablebetween the induction and no-induction groups. Treatment with AMPthrough a transwell did not produce AMP particle settlement on themonolayer. Cells were elongated and spindle-like, with spindle ringformation along the well edge. Like the positive control, the monolayercenter stained a maroon color, and ARS dye concentrated in the spindlering with an intense crimson red color. GnHCl successfully extracted theARS dye and the coefficient of variation in OD₄₅₀ values ranged from 2%to 15% (FIG. 72B).

Results Summary Morphology/ARS Staining

The negative control cultured in α-MEM w/ 10% FBS for 21 days did notdevelop spindle-like cells or a spindle ring along the edge. Thepositive control, after 20 days of induction with AA,β-glycerophosphate, and melatonin, MC3T3-E1 cells developed spindle-likecells and a spindle ring around the edge of the well (FIG. 72 ). ARScolor and stain pattern of the cell monolayer were different between thenegative and positive controls. The negative control monolayer failed tocollect as much dye as the positive control and showed a uniform lightpink color. The positive control monolayer center stained a maroon colorand the ARS dye concentrated in the spindle ring into an intense crimsoncolor (FIG. 72 ).

Treatment with AMP directly in culture medium obscured observation ofcell monolayer in AMP and AMP with induction groups due to settlement ofAMP particles. However, there was no observation of spindle rings alongthe well edge, and some cells along the edge showed spindle-likemorphology. Openings through the AMP particles showed lack of monolayerunderneath. ARS staining showed no difference in color or patternbetween the AMP only group and the AMP with induction group.

Treatment with AMP through transwells did not produce AMP particles ontop of the monolayer. Cell morphology was similar to the positivecontrol, with spindle-like cells and spindle rings forming. The ARSstaining color and pattern also resembled the positive control, with thebackground monolayer staining maroon and the ARS stain concentrating inthe spindle rings in a crimson red color.

ARS Quantitation

GnHCl was necessary and sufficient in extracting ARS staining frommonolayer. Comparing the controls, there was a statistically significant2-fold increase in OD₄₅₀ from the negative control to the positivecontrol (p < 0.01). Also, there was about a 6-fold increase in OD fromthe negative/positive control and AMP+ induction. There is a 6.5xincrease in surface area from 96-well to 24-well, and this could accountfor the increase in OD. We also conducted ARS staining and quantitationon D20 of culturing, which increased the culturing period by 2 days andcould have also increased mineralization.

AMP alone induced statistically significant 10-fold (p < 0.01) and3-fold (p < 0.01) increase in OD₄₅₀ values from the negative andpositive controls, respectively. Thus, AMP alone was sufficient ininducing and promoting differentiation. AMP with induction slightlydecreased the OD₄₅₀ from AMP alone (p < 0.05), but showed a 3-foldincrease in OD from the positive control (p < 0.01). The inductionmedium hindered differentiation and mineralization.

AMP delivered through transwells with induction showed an OD that was3-times lower than AMP delivered directly with induction (p < 0.01), andit showed a slightly lower OD than the positive control (p < 0.05). AMPrequires direct contact to promote differentiation. Without directcontact, AMP inhibits MC3T3-E1 mineralization.

Example 40: Effect of AMP on Induction of Osteogenesis in MSCs

Our results have shown that AMP promotes MC3T3-E1 differentiation whenin contact with the pre-osteoblasts. However, it was unclear how AMPaffects the growth and differentiation of less differentiated and lesscommitted cell lines to the osteoblastic lineage, such as Mesenchymalstem cells (MSCs).

MC3T3-E1 cells are pre-osteoblasts, unipotent, and therefore requireonly supplements to push along their differentiation towardsosteoblasts. Other progenitor cell lines such as embryonic stem cells(ESCs) or mesenchymal stem cells (MSCs) are less differentiated and areoligopotent and pluripotent, respectively. Thus, by studying the effectof AMP on MSCs derived from different areas of the human body, we canbetter understand the role of AMP in osteoblast differentiationprogramming and the factors involved. This investigation would allow usto narrow down which cell types AMP can affect and what effects it hason inducing osteogenesis in different progenitor cells.

Experimental Design

The following cell lines were used: MC3T3-E1 (ATCC, Manassas, VA),Mesenchymal stem cells derived from human bone marrow cells (Lonza,Wlakerfield, MD), Limbal Niche cells (Tissue Tech, Miami, FL), humanAmniotic Membrane (hAM) stromal cells (Tissue Tech, Miami, FL), andhuman umbilical cord vein endothelial cells (HUVEC) (ATCC, Manassas,VA).

Cells were seeded at 3×10⁴ cells/cm²/well in αMEM medium plus 10% FBS in96-well plastic culture plates. Upon confluence, cells were induced toosteoblast differentiation by adding ascorbic acid, β-glycerolphosphate,and melatonin (AGM). For each condition, N=5 was tested. Total inductiontime = 20 Days. Day 0 counted as the day of cell seeding, and inductionfollowed after cell confluence. Each cell type had 3 experimentalgroups: negative control, positive control, and AMP treatment only. ForAMP treatment, an AMP Concentration of 125 µg/ml was used. The medium(100 µL) was changed every 3 days for culture time of 20 days. Anegative control with AMP (without induction or cells) was added. ARSstaining and quantitation were performed as described above on D20.Extracts were then read at 450 nm.

Results

HUVEC cells formed a net-like pattern of cell growth by Day 4 (FIG.74A). However, there was significant cell death with dead cells settledon top of the network of cells until Day 21. Most of the HUVEC cellscould not be fixed with 10% Paraformaldehyde and the few cells stainedwith ARS showed a dark brown color. Although AMP settled on top of theHUVEC cells and covered the network of cells, the AMP particles detachedfrom the plastic well with the cells upon ARS staining; the fewremaining AMP particles also stained a dark brown.

hBM MSCs, without induction, maintained a long fibroblastic shape (FIG.74A). With induction, MSCs became elongated with more raised cell edgesby Day 4. By Day 10, induced MSCs developed spindle-like cells, andcells grew overlapping layers with each other on the monolayer. On Day17, the overlapping spindle cells formed a dense ring about 5 mm fromthe center of well. ARS staining showed that the un-induced MSCsmonolayer stained a cream color, and the spindle ring stained ared-orange color. AMP-treated MSCs contained AMP particles that coveredthe monolayer. With time, the monolayer retracted around concentratedareas of AMP particles. ARS staining showed a deep red-brown color.

hAM stromal stem cells, without induction, maintained a rectangularshape (FIG. 74A). By Day 4, with induction, cell morphology changed andcells elongated with some developing fusiform shapes. AMP particlessettled and covered some of the monolayer by Day 4 in AMP-treated stromacells. The cells not covered by AMP particles in Day 4 were rectangularin shape. By Day 17, cells not covered by AMP particles were elongatedsimilar to induced cells in the positive control. By Day 21, AMPparticles covered the well and cell morphology could not be observed.ARS staining showed the un-induced cells staining a cream color, whilethe induced cells stained a light pink color. The AMP-treated cellsstained a deep red-brown color similar to the AMP-treated hBM MSCs.

The extract with 4M GnHCl yielded the coefficient of variation in OD₄₅₀values ranged from 2% to 15% (FIG. 74B).

Results Summary

The results indicated that no mineralization was noted in the negativecontrol of HUVEC with either inductive agent or AMP. For both hBM MSCand hAM stromal stem cells, mineralization was promoted by the inductiveagent, which was less than that promoted by AMP.

Example 41: Effect of AMP on Mineralization and Cell ProliferationDuring MC3T3-E1 Differentiation

MC3T3-E1 cells undergo three main stages before becoming a matureosteoblast: proliferation, matrix deposition/maturation, andmineralization (FIG. 75 ). Our results have shown that AMP promotesmineralization in MC3T3-E1 cells. In viewing the ARS staining, it showsthat cells treated with AMP stained a darker and denser (FIG. 72 ) thanthe positive and negative controls. Additionally, there was a lack ofmonolayer underneath the AMP particles (FIG. 77 ) when the AMP particlescovered the monolayer. One possibility was that AMP is acting as ascaffold for MC3T3-E1 cells and this interaction in the 3D matrixallowed cells to grow and mineralize. Thus, AMP may be promotingmineralization by increasing proliferation of MC3T3-E1 cells.

Experimental Design

MC3T3-E1 cells were seeded at 3×10⁴ cells/cm²/well in 24-well with αMEMmedium plus 10% FBS as described above. Upon confluence, cells wereinduced to differentiation by adding ascorbic acid, β-glycerolphosphate,melatonin. For each condition, N=3 was tested. Day 1 counted as the dayof cell seeding, and induction followed after cell confluence. Totalinduction time = 20 Days. Four time-points were sampled: D1, D2, D7,D10, D13, D20. Each time-point had four groups: negative control,positive control, AMP treatment only, AMP + (w/ induction). The AMPconcentration employed was 125 µg/ml.

For the proliferation assay measured by MTT assay, the culture periodwas 9 days for proliferation. 4 timepoints were sampled: D1, D2, D4, andD9. Each time-point had 3 groups: cells only, AMP only, AMP + cells. TheAMP concentration employed was 125 µg/ml.

Results Mineralization

On Day 1, cells were seeded for 24 hours and were not treated withinduction medium or AMP (FIG. 76 ). Cells were round and appear moreraised on the monolayer. ARS staining showed a light beige color withlittle to no mineralization. On Day 2, without induction, negativecontrol cells became hexagonal. ARS stained the monolayer a light peachcolor. With induction, positive control cells looked identical tonegative control with little mineralization and ARS staining a lightpeach color. Treatment with AMP caused MC3T3-E1 cell morphology tochange, and fusiform shaped cells and spindle cells were observed. ARSstaining showed increase in mineralization from Day 1 and the monolayerstained a light pink color. The area where AMP particles settled,however, stained a reddish-brown color. AMP treatment with inductionalso changed some cell morphology. Fusiform shaped cells were presentand the monolayer also stained a light pink color with areas around AMPparticles staining reddish-brown. On Day 7, negative control cellsappeared more hexagonal and the cell boundaries are more defined. Thereis an increase in mineralization and the monolayer stained a light beigecolor. The positive control, with induction, developed spindle shapedcells and spindle rings. ARS also stained a deep pink color with moremineralization. Treatment with AMP and AMP with induction caused AMPparticles to settle and cell morphology could not be viewed. A cellmonolayer with individual cells could not be seen. ARS showed areasaround AMP particles staining a red-brown color and the monolayerstaining a light pink. GnHCl successfully extracted the ARS dye and thecoefficient of variation in OD₄₅₀ values ranged from 6% to 16%.

Results Summary

Negative control MC3T3-E1 cells show increase in ARS staining andtherefore mineralization with increasing cell culturing after Day 2(FIG. 76 ). Similar to previous results, positive control MC3T3-E1 cellscultured with induction medium underwent cell morphology change anddeveloped fusiform shaped cells and spindle rings by Day 7 (FIG. 76 ).After 1 Day of AMP treatment (D2), with or without induction, a changein cell morphology could be seen. Cells become spindle and evenfibroblast shaped (FIG. 76A). This change was not observed in thenegative or positive control. Additionally, this change was observed ininduced MC3T3-E1 cells after at least 4 days of culturing (FIG. 76A).

AMP treated and AMP with induction treated cells showed statisticallysignificant increase in mineralization from the positive control by Day2, as shown through ARS quantitation (FIG. 76B). The monolayer stained alight pink color, but small areas where AMP particles settled showedincreased mineralization and stained a deep red-brown color (FIG. 76A).This increase in mineralization continued throughout the culturingperiod.

There was no increase in promoting mineralization with AMP only whencompared to AMP with induction treatment.

Proliferation

To determine whether AMP promoted mineralization via promoting cellproliferation, MC3T3-E1 cells were seeded at 3×10⁴ cells/cm²/well in96-well with αMEM medium plus 10% FBS. Upon confluence, the AMP groupwas treated with fresh 125 µg/ml AMP added every 3 days in the culturemedium. The MTT assay was conducted on Day 1, 2 and 4, while the BrdUassay was conducted on Day 1, 2, and 16.

In untreated MC3T3-E1 cells, cell viability increases from Day 1 to Day4 (FIG. 77A). In AMP-treated cells, cell viability decreased on Day 2and then more than doubled on Day 4, following the trend of the cellsonly group. BrdU assay showed decrease in cell proliferation followingDay 1 in both the cells only group and AMP-treated cell group(FIG. 77B).Cell proliferation in the cells only group decreased by more than halfby Day 2 and continued to decrease on Day 16. In AMP-treated cells, cellproliferation showed a statistically significant decrease only by Day16. These findings suggest that AMP did not promote proliferation duringthe culturing period of 16 days.

Results Summary

Cell viability increased in MC3T3-E1 cells from Day 1 until Day 4 asshown through MTT assay. AMP treated MC3T3-E1 cells showed a decrease incell viability from Day 1 to Day 2 but followed an upwards trend likethe untreated cells. However, cell viability on day 4 in the AMP-treatedcells were around half of the untreated MC3T3-E1 cells. Cellproliferation, as measured through BrdU, decreased from Day 1 all theway to Day 16. Unlike the AMP-treated cells, cell proliferationdecreased by more than half by Day 2 in the untreated MC3T3-E1 cells. ByDay 16, AMP-treated and untreated MC3T3-E1 cells exhibited the samelevels of cell proliferation.

Example 42: Identification of Genes Expressed During the Early Stages ofOsteogenesis in AMP-Treated MC3T3-E1 and hBM MSCs

Human bone marrow mesenchymal stem cells (hBMMSCs) are multipotent andcan differentiation into multiple tissue types such as osteoblasts,chondrocytes, and adipocytes (Born, 2012 J Cell Biochem.113(1):313-21.). When transplanted in vivo, they are capable of formingnew bone, and in vitro, hBM MSCs can be directed towards Osteogenesis bycultivating in β-glycerophosphate, ascorbic acid, vitamin D₃, and lowdoses of dexamethasone. Osteogenesis for hBMMSCs is regulated by theexpression of osteoblast-associated genes, including specifictranscription factors, adhesion molecules and proteins of the ECM (Born(2012) J Cell Biochem. 113(1):313-21;Vater (2011) Acta Biomater.7(2):463-77. ). The progression to mature osteoblast mirrors that ofMC3T3-E1 and occurs with the loss of cellular expansion capacity, theincrease of osteogenic markers expression, and the mineralization of theECM (Born (2012) J Cell Biochem. 113(1):313-21). First, cells initiatethe synthesis of the ECM with expression of collagen I (Col I).Simultaneously, bone-specific alkaline phosphatase (bALP) expressionincreases and by Day 4, significant increase in ALP levels in theinduced cells (6x10⁴cells/60mm culture dish) from the control could beobserved (Born (2012) J Cell Biochem. 113(1):313-21; Jaiswal (1997) JCell Biochem. 64:295-312). As differentiation continues, cells produceproteins such as bone sialo protein (BSP), Osteopontin, Osteonectin andosteocalcin. Finally, mineralization of the ECM, much like osteogenesisin MC3T3-E1 cells, indicates a mature osteoblast.

In this example, genes and transcription factors expressed in the earlystages of Osteogenesis in three cell lines: MC3T3-E1 cells and hBM MSCcells were determined. Our results have shown that a statisticallysignificant increase in mineralization could be seen in AMP-treatedMC3T3-E1 cells compared to the positive control by Day 2 (Day 1 oftreatment). Therefore, the experiment focused on the early stages ofOsteogenesis to identify AMP’s effect on specific genes after treatment.

Experimental Design

MC3T3-E1 or human Bone Marrow MSC cells were seeded at 3×10⁴cells/cm²/well in 24-well plates with αMEM medium plus 10% FBS asdescribed above. Upon confluence, cells were induced to differentiationby adding ascorbic acid and β-glycerolphosphate. For each assay at eachtimepoint, N=2 was tested. There were 4 time-points sampled: D0 (afterconfluence but before induction/treatment), D1, D2, D4, and D6. Eachtime-point had three groups: negative control, positive control, AMPtreatment only. AMP concentration used was 125 µg/ml.

Results hMSC Expression

AMP induces robust endogenous expression of BMP2 and BMP6 transcriptswithin 24 hrs of culturing (60 and 5 folds, respectively) (FIG. 78A).BMP2 reached its peak at D1 (120 folds) and maintained a high level(between 60 to 10 folds) until D4 before showing a decline. In contrast,BMP6 peaked at 4 h and then showed a gradual decline from D1 on. As acomparison, AMP did not change expression level of BMP4, BMP7 and BMP9.Nonetheless, AG induced a mild upregulation of BMP2 and BMP6 (1-2 fold)only at D4 and notable upregulation of BMP4 (4-10 folds) after D1. As acomparison, AGM did not change expression of BMP7 and BMP9 either.

Runx2 peaked at D1 for AMP but D2 for AGM (FIG. 78A). Both groups thenhad a gradual decline followed by another peak at Day 6. Runx2 inducesthe differentiation of multipotent mesenchymal cells into immatureosteoblasts, directing the formation of immature bone. Furthermore,Runx2 triggers the expression of major bone matrix genes during theearly stages of osteoblast differentiation, but Runx2 is not essentialfor the maintenance of these gene expressions in mature osteoblasts(Komori (2010) Adv Exp Med Biol. 658:43-9).

Both ALP and Sox9 peaked at D4, with the level upregulated by AMP beingless than AGM (FIG. 78A). This trend is consistent with the view thatALP and Sox9 are downstream of Runx2. ALP is expressed in the earlyphase of osteogenesis and creates an alkaline environment which causescalcium to come out of solution and crystallize.

AMP upregulated VEGF which peaked at 4 h and D4 but AGM upregulated VEGFonly at D4 (FIG. 78A). AMP upregulated CXCR4 while AGM upregulated SDF1at D1, with a rapid decline for the former but a slower one for thelatter. Kortesidis et al. ((2005) Blood. 105(10):3793-801) suggest thatSDF-1 may act to localize primitive uncommitted BMSSC populations withintheir perivascular niche until required to proliferate and differentiatein response to environmental cues that may act to disrupt SDF-⅟CXCR4interactions. In 7 preconditioning experiments of MSCs, expression ofCXCR4 is normally around 2 to 4 fold increase Cencioni et al. ((2012)Cardiovasc Res. 94(3):400-7) however our results with AMP showed a 60fold increase after Day 1.

SDF-1 expression was very high in all groups of the MSCs, although itdoes not appear so in FIG. 78A. SDF-1 was seen around Cycle 20 whereasGAPDH was seen around Cycle 17. From the literature it is known stromalcell-derived factor-1 activates adhesion molecules on progenitor cells,and mAb against SDF-1 inhibits transendothelial migration ofhematopoietic progenitor cells (Imai et al. (1999) Blood 93(1):149-56).SDF-1 activates CXCR4+/CD34+ cells and leads to their adhesion andtransendothelial migration (Bhakta et al. (2006) Cardiovasc Revasc Med.7:19-24.). GAPDH expression did increase in the AMP group from Day 2 toDay 6, therefore one would assume proliferation. This trend was not seenin the other samples. Furthermore, BMP9 was not detectable (after Cycle40), CXCR4 was detected around Cycle 33, VEGF was detected around Cycle19, and SOX-9 was detected around Cycle 27. BMP4 was detected aroundCycle 28, BMP7 was not detected (after Cycle 40), BMP2 was detectedaround Cycle 20, Runx2 was around Cycle 25, BMP6 was detected aroundCycle 27, and ALPL was detected around Cycle 24.

MC3T3-E1 Expression

It is known that MSC are multipotent as where osteoprogenitors arealready pushed farther down the lineage pathway and should only producebone formation. Contrary from what was expected, BMP2 expression wasbarely expressed in Mouse cells especially compared to human MSC (FIG.79 ). BMPs have been shown to be inefficient in promoting osteogenesisin human, yet are more than capable in Mouse cells (Osyzka et al. (2004)Cells Tissues Organs. 176(1-3):109-19., Skarzynska et al. (2011) ConnectTissue Res. 52(5):408-14). Expression of BMP-2 is not prominent in MC3T3cells as indicated by gene array (Beck et al. (2001) Cell Growth &Differentiation 12: 61-83). The differences seen between human androdent cells may indicate that, unlike Smad1 in human cells, rodentSmad1 does not undergo ERK linker phosphorylation during osteogenesis.Alternatively, Smad1 activity in rodent cells may not be suppressed byERK-mediated linker phosphorylation (Skarzynska et al. (2011) ConnectTissue Res. 52(5):408-14). BMP-induced osteogenesis in poorly responsivehuman MSC requires modulation (inhibiting) of ERK andphosphatidylinositol 3-kinase (PI3-K) pathways; inhibiting theinsulin/IGF-I-activated PI3-K/AKT pathway decreases BMP-induced alkalinephosphatase and osteopontin expression in serum-free cultures of humanMSC, but increases BMP activation of Smads (Osyzka et al. (2005)Endocrinology. 2005 (8):3428-37).

Runx2 also was shown to be different in the MC3T3 cells compared tohMSCs. There is clearly an incident on Day 2 that causes an upregulationof genes in MC3T3 cells. The AGM groups appeared to have oppositeeffects of the AMP group, which may be related to Pi3K, MAPK pathways.Ibsp, also known as Bone Sialprotein (BSP), is upregulated on Day 2 inAMP but not expressed other times. In the positive group, BSP is highlyexpressed after Day 2.

Bglap-rs1, also known as Osteocalcin (OCN), is also upregulated on Day 2in AMP but not expressed elsewhere. Consistent with BSP, OC is highlyexpressed after Day 2 in positive group. Through analysis of theseresults one would think BSP and OCN need to be upregulated since theyare known to be expressed by mature osteoblasts. Runx2 regulates theexpression of Col1, BSP and OCN in MC3T3, which is consistent with ourresults. Runx2 increases on Day 2, as does BSP and OCN, and is notexpressed elsewhere. In summary, AMP downregulates osteogenic geneexpression but promotes mineralization.

Example 43: Effect of nHC-HA/PTX3 Purified From AM on MineralizationUsing MC3T3-E1 Model System

Previous experiments have clearly demonstrated AMP’s unique propertiesto promote mineralization independent of inducing agents such asascorbic acid, β-glycerol phosphate, and melatonin (AGM). In theseexperiments, AMP was added to the murine osteoblast progenitor cell line(MC3T3) in the presence or absence of AGM to determine the potency ofthe AMP. Alizarin Red Staining with the GnHCl extraction method was alsoused at multiple time points for further quantification. It wasconcluded AMP not only enhanced mineralization independent of AGM, butalso enhanced mineralization at a faster rate. Further investigation ofAMP showed that the osteoinductive effect exhibited by AMP requiresdirect cell contact. That is, under direct contact with AMP, cells willhave accelerated and enhanced mineralization without the introduction ofAGM.

Previous studies showed that MC3T3-E1 differentiation under inductioncan be subdivided into three stages, i.e., proliferation (day 1 to 9),ECM formation (day 9 to day 16), and mineralization (deposit minerals informed ECM) (day 16+) (Quarles et al. (1992) J Bone Miner Res.7(6):683-92.; Hong et al. (2010) Exp Cell Res. 316(14):2291-300).However our previous Aim 4 has shown mineralization can be easilydetected within 7 to 10 days using AMP. Therefore we will perform ourARS assay at day 8 in order to determine if nHC-HA/PTXS purified from AMis responsible for promoting mineralization. Day 8 was picked becauseresults should be noticeable as seen in Aim 4 and the media will onlyhave to replaced on day 0, day 3 and day 6.

It is already known that nHC-HA/PTX3 is responsible for amnioticmembrane’s known anti-inflammatory, anti-angiogenic and anti-scarringtherapeutic actions. It is our hypothesis that immobilized HC-HC/PTX3 isresponsible for AMP’s effect on promoting mineralization. Because italso contains HA, we will also compare nHC-HA/PTX3 to HA to see theputative effect is uniquely present in nHC-HA/PTX3 but not in HA.Hyaluronan (HA) is an unsulfated glycosaminoglycan consisting of asingle repeating disaccharide unit. It is an important component inconnective tissue promoting matrix assembly and tissue hydration. Lubenet al speculated HA acts as a calcium binding agent to act as a barrierto the diffusion of enzymes away from the resoprtion site or to regulatethe mobility of osteoclasts. Stern and Raisz stated “hyaluronic acidseems to be the most appropriate to study because it has been clearlylinked to bone resorption. By the nature of its hygroscopic propertiesHA can occupy 10,000 times its own volume. Thus, HA allows proliferatingcells to avoid inhibitory contacts. Hyaluronic acid synthesis precedesmitosis and dissociates the dividing cell from its substratum,permitting cell movement (Balazs (2001) Am J Physiol RegulatoryIntegrative Comp Physiol 280: R466-R472).

Experimental Design:

Murine MC3T3-E1 cells (C-136) were taken from liquid nitrogen freezerand grown on 100 mm dish (five dishes) in αMEM medium (10 ml per 100 mmdish) plus 10% FBS changed every 3 days till 80% confluence ~1.5 ×10⁶cells [4*(3.1×10⁴)*9= 1,116,000 cells]. Cells were then seeded at3.1×10⁴ cells/cm²/96 plastic well. Medium (100 ul per 96 well) wasreplaced every 2-3 days, i.e., at Day 0(Wed), 2(Fri), and 5(Mon) andcultures will be terminated at Day 8. N=4 was tested per condition.

A summary of the Groups was as follows:

-   Control Groups:    -   Negative Control: Nothing on conventional 96 well plate    -   Positive Control 1: AGM on conventional 96 well plate    -   Positive Control 2: 125 µg/ml of AMP added every 3 days on        conventional 96 well plate-   Experimental Groups:    -   Negative Control: Covalink-NH 96 Well Plate    -   Experimental Group 1: AGM added every 3 days added to        Covalink-NH 96 Well Plate    -   Experimental Group 2: 20 µg/ml of HA immobilized on Covalink-NH        96 Well Plate    -   Experimental Group 3: 20 µg/ml of HA immobilized on Covalink-NH        96 Well Plate with AGM added every 3 days (H-124)    -   Experimental Group 4: 20 µg/ml of nHC-HC/PTX3 immobilized on        Covalink-NH 96 Well    -   Experimental Group 5: 20 µg/ml of nHC-HC/PTX3 immobilized on        Covalink-NH 96 Well Plate with AGM added every 3 days

For the AGM groups: On Days 0 and 3, Osteogenesis induction media #1(ascorbic acid, β-glycerolphosphate) was replaced. On Day 6,Osteogenesis induction media #2 (ascorbic acid, βglycerolphosphate,melatonin) was replaced. On Day 0, only 0.2 ml of 10X Induction mediawas made. On Days 3 and 6, 10 ml of the Osteogenesis induction media wasprepared fresh. Instructions for Induction Medias obtained from In VitroOsteogenesis Assay Kit (Millipore).

Induction media #1: 9.88 ml of αMEM medium plus 10%, 20 µl Ascorbic Acid2-Phosphate 500X (Millipore, Part. 2004011), 100 µl Glycerol 2-Phosphate100X (Millipore, Part. 2004011).

Induction media #2: 9.87 ml of αMEM medium plus 10%, 20 µl Ascorbic Acid2-Phosphate 500X (Millipore, Part. 2004011), 100µl Glycerol 2-Phosphate100 X (Millipore, Part. 2004011), 10 µl Melatonin 50 uM (Millipore,Part. 2004011). Add 500 ul dH20 to 6 ug of melantonin supplied.

ARS Staining and Quantification was performed as described above.Pictures were taken at 10X using Nikon Eclipse CFI60.

Results

The MC3T3-E1 cells were cultured on the different well plates for 8days. On Day 8, phase contrast pictures were taken of the wells and canbe seen below (FIGS. 79A, 79B). Negative control wells showed roundcells and the ARS stained a very light pink. Wells with induction mediashowed much brighter red color and spindle cells were seen on theperiphery of the wells. The ARS staining was seen more in abundance inthe outer periphery ring. Treatment with AMP showed a crimson red afterARS treatment and the cells were rather hard to see because the AMPsettled on top of them. No aggregation was seen during the experiment(even though aggregation has been seen with other cell types onimmobilized HC-HA.) Microscopy pictures were taken on Days 6, 7 and 8.

Guanidine hydrochloride extraction method was used on Day 8 and the wellplates were incubated overnight. GnHCl was able to extract the ARS dyealthough it was hard to tell by the naked eye; all the wells seemed tohave the same light pink/red color. The ARS extraction was quantified at450 nm because the plate reader did not have the capabilities of readingat 490 nm or closer. Results can be seen in FIG. 79C. The * symboldenotes statistical significance of p<0.05. (+ denotes with AGM, -denotes without AGM)

In agreement with previous results, AMP was able to successfully promotemineralization without the need of induction agents. This experimentonly lasted 8 days so the results aren’t as noticeable as in Aim 4 whichlasted 20 days. All conditions that were treated with AGM showed anincrease in mineralization from their negative control counterpart. Ourresults also show that immobilized nHC-HA/PTXS is not responsible forpromoting mineralization in AMP. Therefore there must be another activecomponent of AMP that is promoting mineralization.

Example 44: Effect of HC-HA/PTX3 (PBS) and HC-HA/PTX3 (Gn) onEndochondral Ossification

Master transcription factors for osteogenesis and chondrogenesis (Runx2and Sox9, respectively) were expressed by cells in both HC-HA conditionsthrough the 14 day culture period. HC-HA/PTX3, both soluble andinsoluble, were able to promote the expression of BMP2 and to an extentBMP6 without osteoinductive agents AGM (i.e., ascorbic acid,glycerolphosphate, melatonin) commercially provided (see below).

Chondrogenic marker Collagen 2 was highly expressed by HC-HA/PTX3 (PBS)without the need for AGM. HC-HA/PTX3 (Gn) with the addition of AGM alsowas able to upregulate Collagen 2. Osteogenic markers (BSP, ALPL, Osx)were upregulated by the HC-HA conditions on Day 14 thus confirming atransition from a cartilage to bone genotype.

Experimental Design

Culture conditions: Human bone marrow-derived mesenchymal stem cellspurchased from Lonza (Basel, Switzerland) were taken from liquidnitrogen freezer and grown on 100 mm dish in the same medium changedevery 3 days till 80% confluence. Cell culture medium was αMEMcontaining 10% fetal bovine serum and antibiotics. Culture medium (10mlper 100 mm dish) was changed every 3 days, and cells subpassaged at 80%confluence until they reach the above desirable cell numbers. For theexperiment the cells were seeded at 3.1x10⁴ cells/96 plastic well onimmobilized HA, HC-HA (PBS) or HC-HA (Gn) with or without osteoinductiveagents Ascorbic Acid 2- Phosphate, Glycerol 2-Phosphate and Melatonin(AGM). The final concentration of AGM added was 0.2 mM, 10 mM and 50 nM,respectively. AGM was added simultaneously when cells were seeded (D0)and mRNA was extracted at Days 1, 7 and 14. In order to quantify geneexpression, qPCR was performed. Culture medium (100 ul per 96 well) wasreplaced every 3 days.

A summary of the experimental Groups was as follows:

-   Negative Control: Covalink-NH 96 Well Plate-   Experimental Group 1: AGM added every 3 days added to Covalink-NH 96    Well Plate-   Experimental Group 2: 20 µg/ml of HA immobilized on Covalink-NH 96    Well Plate-   Experimental Group 3: 20 µg/ml of HA immobilized on Covalink-NH 96    Well Plate with AGM added every 3 days-   Experimental Group 4: 20 µg/ml of 4X nHC-HC/PTX3 immobilized on    Covalink-NH 96 Well Plate-   Experimental Group 5: 20 µg/ml of 4X nHC-HC/PTX3 immobilized on    Covalink-NH 96 Well Plate with AGM added every 3 days-   Experimental Group 6: 20 µg/ml of 4X nHC-HC/PTX3 (GuHCl extraction)    immobilized on Covalink-NH 96 Well Plate-   Experimental Group 7: 20 µg/ml of 4X nHC-HC/PTX3 (GuHCl extraction)    immobilized on Covalink-NH 96 Well Plate with AGM added every 3 days

For the AGM induction groups: On Days 0 and 3, Osteogenesis inductionmedia #1 (ascorbic acid, glycerolphosphate) replaced the media. On Day6, Osteogenesis induction media #2 (ascorbic acid, glycerolphosphate,melatonin) will replace the media. On Day 0, 10X Induction media wasmade. On Days 3 and 6, 10 ml of the Osteogenesis induction media wasprepared fresh. Instructions for preparation of Induction Media obtainedfrom In Vitro Osteogenesis Assay Kit (Millipore).

Induction media #1: 9.88 ml of αMEM medium plus 10%, 20 µl Ascorbic Acid2-Phosphate 500X (Millipore, Part. 2004011), 100 µl Glycerol 2-Phosphate100X (Millipore, Part. 2004011)

Induction media #2: 9.87 ml of αMEM medium plus 10%, 20 µl Ascorbic Acid2-Phosphate 500X (Millipore, Part. 2004011), 100 µl Glycerol 2-Phosphate100 X (Millipore, Part. 2004011), 10 µl Melatonin 50 uM (Millipore,Part. 2004011). Add 500 ul dH20 to 6 ug of melantonin supplied.

mRNA was extracted from cells on Days 1, 7, and 14 and gene expressionwas determined by QPCR (FIGS. 80A-E). The following genes were assayed:Osteogenesis markers Runx2, alkaline phosphatase (ALPL), markersCollagen 1 (COL1), Osterix (OSX) and Bone Sialoprotein (BSP) andchondrogenesis markers Sox9 and Collagen 2 (COL2), hypertrophic markersCollagen 10 (COL10) and MMP13. ARS staining and quantification wasperformed on Day 14 cultures as described above (FIGS. 81A, 81B).

AGM upregulated BMP4 on plastic. HA upregulated BMP4 (early) butdownregulated (late) BMP6 and did not affect BMP2 (FIG. 80B). Noliterature data suggests HA itself upregulates BMP. However, addition ofAGM upregulated BMP2 and BMP6.

4X Soluble HC-HA initially upregulated BMP4 but downregulated BMP4 late(like HA) and markedly upregulated BMP2 (like AMP, but without transientBMP6) (FIG. 80B). In contrast, addition of AGM did not change theexpression pattern of BMPs.

4X Insoluble HC-HA initially upregulated BMP4 but downregulated BMP4(late) (like HA) and markedly upregulation of BMP2 (like Soluble HCHA)(FIG. 80B). [Identical to Soluble] Similarly, addition of AGM did notchange the expression pattern.

Our results show soluble HC-HA and insoluble HC-HA were able to formbone differentiation and mineralization through an endochondralmechanism. Expression of bone markers (Col1, Osx, ALP, and BSP) wereapparent as were expression of chondrocyte markers (Col2) andhypertrophy markers (Col10, MMP13) (FIGS. 80A-E). The difference betweenthese HC-HA conditions is that insoluble HC-HA was able to promotegreater amplitude of gene expressions and more noticeable bone nodules(even without AGM) while soluble HC-HA requires AGM (data not shown).Thus, HC-HA/PTX3, both soluble and insoluble, were able to promote theexpression of BMP2 without osteoinductive agents AGM.

HA without AGM also showed chondrogenic markers (COL2) but also showedsigns of bone formation with ARS defined mineralization and a slightincrease in ALP and OC. However, HC-HA/PTX3 (PBS) had greaterchondrogenic expression and higher expression of bone markers ALP, Osxand BSP than HA. Yet neither of these conditions expressed significanthypertrophic markers. HC-HA/PTX3 (Gn) expressed much greater expressionof ALP, OSX and BSP than the two aforementioned conditions. Hypertrophicmarker MMP13 was also expressed as was slight expression of chondrogenicmarker COL2.

HA plus AGM promotes osteogenesis with increased BMP2, ALP, Osx, BSP andOC expression and exhibits hypertrophic markers COL10 and MMP13.However, HA produced less bone specific mRNA expression and bone noduleformation than HC-HA groups. Another key difference was HA downregulatedSOX9 but increased BMP6 expression late.

All prior data indicates insoluble HCHA is the strongest inducer of boneand, more importantly, induces an endochondral mechanism.

Example 45. nHC-HA/PTX3 Suppresses Inflammatory and Immune Responses andImproves Murine Corneal Allograft Survival

Experimental and clinical studies have shown that amniotic membrane(AM), AM extract, and nHC-HA/PTX3 [a covalent complex formed by heavychain (HC) of inter-α-trypsin inhibitor (IαI) and hyaluronan (HA)]suppress pro-inflammatory responses. This example demonstrates thatnHC-HA/PTX3/PTX3 can regulate T cell responses and reduce murine cornealallograft rejection.

T cell activation may be assessed by proliferation and production ofvarious cytokines (FIG. 82 ). In this instance, splenocytes wereisolated from OT-II mice that express a transgenic TCR specific forovalbumin (OVA), and stimulated with OVA up to 4 days (FIG. 83 ). Cellproliferation was measured by BrdU labeling and expression of cytokines(IFN-γ and IL-2) was measured by the respective ELISA. nHC-HA/PTX3 butnot HA at 1 mg/ml significantly suppressed the proliferation (FIG. 84 )and production of IFN-γ and IL-2 (FIG. 85 ) in splenocytes with OVApeptide at day 2 and day 4 (all p < 0.05). Furthermore, corneal T cellswere activated in vivo by LPS injection.

Optimization of injection sites, volume, and frequency with nHC-HA/PTX3before or during intracorneal injection of LPS was determined by influxof EGFP-positive macrophages into corneas of Mafia mice. The injectionregimen was further optimized by giving 5 µl at each injection betweensubconjunctiva and fornix to all four quadrants. At day 4 afternHC-HA/PTX3 treatment, corneas were digested with 820 units/ml ofcollagenase at 37° C. for 1 h. EGFP- and EGFP+ cells were isolated byFACS. Pretreatment of nHC-HA/PTX3 3 days prior to LPS injectionsignificantly suppressed the influx of EGFP+ macrophages to LPS-insultedcorneas (9.1±0.3 vs. 12.3±0.4, nHC-HA/PTX3 vs PBS, p =0.02) (FIG. 86 ).Importantly, even though EGFP+ macrophages did migrate into corneas,some of them were polarized into M2 phenotype as suggested bysignificant up-regulation of Arg-1 and IL-10 but down-regulation ofIL-12 (p < 0.05) (FIG. 87 ). mRNA expression of Arg-1, IL-10, and IL-12were measured by qPCR. Finally, allogenic corneal transplantation wasperformed using wild-type BALB/c mice as recipients and C57BL/6 mice asdonors, and its outcome scored by graft clarity measured twice a weekusing slit lamp biomicroscopy. Grafts that received two consecutivescores ≥ 3 without resolution were considered rejected. Compared to PBScontrol, allograft rejection was significantly suppressed by injectionof 10 µl nHC-HA/PTX3 at one quadrant twice a week (p < 0.05), andfurther reduced by injection with 5 µl at 4 quadrants twice a week (p <0.002) (FIG. 88 ).

These experiments demonstrate that nHC-HA/PTX3 significantly suppressesmurine corneal allograft rejection. The mechanism of this action may becontributed by nHC-HA/PTX3′s ability to down-regulate pro-inflammatorymacrophages and to suppress T cell immune response.

Example 46: Treatment of Mouse Dry Eye Caused by Desiccating Stress bynHC-HA/PTX3 and AMP

Dry eye, also known as dysfunctional tear syndrome, is a common ocularsurface disease with high prevalence and significant morbidityworldwide. It is an autoimmune-based inflammatory disease characterizedby chronic auto-reactive T cell-mediated inflammation and dysfunction ofthe lacrimal function unit (LFU; cornea, conjunctiva, lacrimal glands,and meibomian glands). Sjögren’s syndrome (SS) is a prevalent chronicautoimmune disorder characterized by infiltration of salivary andlacrimal glands by mononuclear cells, causing secondary destruction ofthe parenchymal tissue.

Keratoconjunctivitis sicca (KCS) in SS is a severe and potentiallysight-threatening ocular surface epithelial disease characterized byinfiltrating CD4+ T cells producing IL-17 and interferon (IFN)-γ.Compounds that inhibit T cell activation (e.g., cyclosporine A)attenuate dry eye disease in both animals and humans. Macrophages mayundergo classical M1 activation (e.g., by IFN-γ and/or TLR ligands suchas LPS) to express high levels of proinflammatory cytokines (such asTNF-α, IL-12, and IL23), which activate Th1 and Th17 lymphocytes (FIG.89 ) leading to many chronic inflammatory diseases. This exampledemonstrates that nHC-HA/PTX3 and AMP administration may be useful inthe treatment of such conditions.

Infiltration of Macrophages Into Corneas is Inhibited With FourInjection Sites for Each Eye

MAFIA mice permit in vivo tracking of macrophage influx as they arelabeled with EGFP. These mice were used to determine if nHC-HA/PTX3 orAMP prevents LPS-induced macrophage influx to the cornea, a model forkeratitis. LPS was injected between subconjunctiva and fornix at asuitable volume of 5 µl or less at each injection site. Eyes of MAFIAmice (macrophages are EGFP+) were intrastromally injected with LPS (5 µgper eye). In each eye, OS was treated with PBS (2 or 4 injection sites)while OD was treated one time with either nHC-HA/PTX3 (2 or 4 injectionsites; 5 µl of 1 mg/ml HA in nHC-HA/PTX3 per injection site) or AMP (2or 4 injection sites; 5 µl of 10 mg/ml protein in AMP per injectionsite). The treatment was immediately after LPS injection. Images ofwhole corneas were taken with in vivo intravital microscopy on day 1,day 2, day 3 and day 6. EGFP-positive cells were counted based on theintensity of green fluorescence.

At Day 1, EGFP-positive macrophages are detected in the most cornealperipheral area after LPS injection with PBS treatment. Treatment witheither nHC-HA/PTX3 or AMP did not significantly increase or decreasemacrophages in corneas.

At Day 2, the macrophages in corneas with PBS treatment increasedsignificantly (p < 0.05) from Day 1, so did with treatment ofnHC-HA/PTX3 (2 and 4 injection sites, p < 0.05) and of AMP (2 and 4injection sites, p < 0.05). Specifically, more macrophages wereinfiltrated in corneas treated with 2 injections of nHC-HA/PTX3 thanthose with PBS treatment (p > 0.05), but less are in corneas with 4injections of nHC-HA/PTX3 (p > 0.05). For AMP treatment, 2 injectionshad no significant effect but 4 injections slightly decreased theinfiltration of macrophages, suggesting 4 injections of eithernHC-HA/PTX3 or AMP for each eye is necessary to have an effect onreducing the infiltration of macrophages.

At Day 3, the infiltration of macrophages continued with treatment ofPBS, nHC-HA/PTX3, and AMP.

There was no inhibition of the infiltration with treatment of either 2injections, 4 injections of nHC-HA/PTX3, or 2 injections of AMP. Theonly treatment shows the inhibition is the 4 injection of AMP (p <0.05).

At Day 6, the infiltration of macrophages decreased. However, notreatment of HC-HA/PTX3 or AMP had a significant inhibitory effectcompared to control.

These data showed EGFP-positive macrophages continue to infiltrate intoLPS-injected corneas from Day 1 to Day 3 and peaks at Day 4 or Day 5,then decline at Day 6. This is consistent with the previous reporteddata (FIG. 90 ). The infiltration of macrophages was slightly inhibitedby treatment with 4 injections of nHC-HA/PTX3 per eye at Day 2 or with 4injection of AMP at Day 2 and Day 3. This suggests AMP has a betterpotency than nHC-HA/PTX3 in blocking the influx of macrophages elicitedby LPS.

Pretreatment With AMP Significantly Inhibits the Macrophage InfiltrationIncited by Injuries Due to Additional Injections if Followed bySubsequent Injections of Either nHC-HA/PTX3 or AMP

The left eye (OS) of each MAFIA mouse was pretreated with PBS (5 µl) orAMP (5 µl of 10 mg/ml protein) at 4 sites of subconjunctival/fornix asdefined above. The right eye (OD) of each mouse was left untreated.Three days later, each eye was injected with LPS (5 µg) to the corneaand immediately followed by treatment with PBS (5 µl), HC-HA/PTX3 (5 µlof 1 mg/ml HA), or AMP (5 µl of 10 mg/ml protein) at 4 sites. Theinfiltration of EGFP⁺ macrophages was counted using in vivo intravitalmicroscopy, which did not disclose any significance (P > 0.05) inreducing macrophage influx to mouse corneas (data not shown). We theninvestigated the accuracy of this quantitative method by in vivofluorescence microscopy.

To measure more accurately the infiltrated macrophages and examine theresultant macrophage phenotype (e.g., M1 vs. M2), we decided toquantitate EGFP-positive macrophages by subjecting the corneas removedat Day 4 to collagenase digestion and FACS. EGFP-positive macrophageswere then normalized by EGFP-negative cells as a ratio to assess theextent of macrophage infiltration. In groups with no pretreatment, LPSinjection caused macrophage infiltration in the PBS control group (FIG.91 , A, the blue bar). This infiltration was significantly inhibited bynHC-HA/PTX3 (9.1±0.3 vs. 12.3±0.4, p=0.02) or AMP (2.1±0.1 vs. 12.3±0.4,p=0.02). AMP treatment was better than nHC-HA/PTX3 treatment ininhibition of the macrophage infiltration (p=0.02).

In groups with pretreatment, LPS injection caused significant macrophageinfiltration in PBS control group compared with no pretreatment(37.2±1.3 vs. 12.3±0.4, p=0.01) (FIG. 91 , A, the red bar). Thisdifference was expected as 4 subconjunctival injections made duringpretreatment caused injury that elicited inflammation, which augmentedmacrophage influx to the cornea which was later on treated by LPS.Nonetheless, this dramatic increased infiltration was completelyinhibited by AMP pretreatment followed with either nHC-HA/PTX3 (8.2±0.3vs. 37.2±1.3, p=0.02) or AMP treatment (2.3±0.1 vs. 37.2±1.3, p=0.02).Again, AMP pretreatment followed by AMP treatment was better than AMPpretreatment followed by nHC-HA/PTX3 treatment in inhibiting macrophageinfiltration (2.3±0.1 vs. 8.2±0.3, p=0.02). However, there was nosignificant difference in inhibition between the group with nopretreatment and the group with pretreatment of AMP for eithernHC-HA/PTX3 or AMP (p >0.05). qPCR data (FIG. 91 , B) show pretreatmentof nHC-HA/PTX3 decreases M1 markers if IL-12p40 and IL-12p35 whileincreases M2 markers of Arg-1. AMP treatment and pretreatmentsignificantly decreases IL-12p40 and IL-12p35 but greatly increasesArg-1 and IL-10. In all, AMP pretreatment can completely eliminate themacrophage infiltration incited by additional injuries during thepretreatment. Such an effect is sustained by subsequent injection ofeither nHC-HA/PTX3 or AMP simultaneously with LPS injection. Thisbenefit is noted 4 days later, and AMP is more potent than nHC-HA/PTX3.

nHC-HA/PTX3 or AMP Can Reduce DS-induced ALKC in a Murine ExperimentalDry Eye Model Design

Species: C57BL/6 mice

Endpoints: corneal epithelial barrier function (OGD staining)

Sample Size: 15 mice per group

Groups: 2 controls and 3 treatment groups (PBS, nHC-HA/PTX3, andAMP): 1) Non-dry eye, untreated control (UT) - kept in a separatevivarium room; 2) Experimental dry eye, untreated control (EDE); 3) PBS;4) nHA-HC/PTX; 5)AMP.

Desiccating Stress (DS)

The desiccating stress model is created by pharmacological cholinergicblockade of tear secretion and exposure to an air draft and low humidityin an environmentally controlled room for 5 days (Monday-Friday). Miceare placed in specially designed perforated cages which consist ofregular mouse cages that have their sides replaced by a wire mesh toallow air flow through the cage. Each cage is placed in front a constantair flow (electrical fan). Lacrimal gland secretion is inhibited bysubcutaneous administration of scopolamine (0.5 mg in 0.2 mL,Sigma-Aldrich) 4 times per day for 5 days (8:30am, 11:30am, 1:30pm,4:30pm). Humidity in the environmentally controlled room is maintainedat ~25-30% relative humidity, which is achieved by 4 portabledehumidifiers and a dehumidifier unit in the ceiling.

Treatment Procedure (5-Day Protocol)

During each experiment, 3 controls are included:

Untreated control, consists of a group of mice that are kept in thevivarium, under relative humidity of 40~70%. These mice are neverexposed to DS nor receive any topical treatment.

Dry eye control, which consists of a group of mice that are placed inthe environmental dry eye chamber but receive no treatment.

Vehicle control, consists of a group of mice that are subjected to DSbut receive PBS.

In addition, two experimental groups are included: nHA-HC/PTX3 and AMP

For injection, we used nHC-HA/PTX3 (containing 1 mg/ml HMW HA) and AMP(containing 10 mg/ml total protein) with PBS as the vehicle control. Allsolutions (PBS, nHC-HA/PTX3, and AMP) were drawn into a tuberculinsyringe with 30 G. The injection locations are subconjunctiva close tofornix (FIG. 92 ). Four (4) injections at 3, 6, 9, and 12 o′clock with 5µl per injection sites were administrated. The diffused solutioncompletely covered the whole peripheral of conjunctiva and caused aminimum conjunctival or globe congestion/swelling (If any, it shoulddisappear in 15 min) which impeded eye closure and corneal surfacebreakdown or inflammation. The injection was administrated at Day 1 andDay 3 (for all reagents), making a total of 2 times. This injectionprotocol is summarized in Table 6.

TABLE 6 Experimental groups and required reagents for nHC-HA/PTX3 or AMPreduction of DS-induced ALKC in murine experimental dry eye model. GroupTreatment # Mice Injection sites x volume x days x eyes x mice 1 UT 15NA 2 EDE 15 NA 3 EDE + PBS 15 4 × 5 µl × 5 × 2 × 15 = 3.0 ml 4 EDE +nHC-HA /PTX3 (1 mg/ml) 15 4 × 5 µl × 5 × 2 × 15 = 3.0 ml 5 EDE + AMP (10mg/ml) 15 4 × 5 µl × 5 × 2 × 15 = 3.0 ml

Measurement of Corneal Staining

On the morning of 5^(th) day, mice received one s.c. dose of scopolamineafter measurement of tear volume. 2 hours after that scopolamine dosecorneal staining was performed using Oregon Green Dextran (OGD-488),which is a conjugated fluorescent dye of a 70 kDa molecular size(Molecular Probes). The procedure consisted of instillation of 0.5 µl ofOGD on the cornea using a glass capillary pipette, 1 minute beforeeuthanasia. Mice were euthanized by inhalation of isoflurane anesthesicgas followed by cervical dislocation. Eyes were then rinsed with 2 ml ofBSS. Excess liquid was carefully blotted from the ocular surface withfilter papers without touching the cornea. Digital images of both eyeswere captured under 470 nm excitation and 488 nm emission wave lengthsusing a Nikon SMZ-1500 stereo microscope with CoolSnap HQ₂ cooled CCDcamera, with an exposure time of 1 second. Both eyes from each animalwere evaluated. The fluorescence intensity in a fixed region of interest(a 1-mm diameter circle) in the central cornea was measured in 3 digitalimages using Nikon Elements software and data is stored in a database(Excel, Microsoft). Results were presented as mean ± standard deviationof gray levels. Results from 3 separate experiments were averaged forstatistical comparisons of groups.

The effects of nHC-HA/PTX3 and lyophilized amniotic membrane powder(AMP) on levels of T helper cell pathway mediators were compared in anexperimental dry eye (EDE) model created in C57BL/6 mice for 5 days.Expression of Th1 (IL-12, IFN-γ and T-Bet), Th-17 (IL-23, IL-17, ROR-γt,IL-6, TGF-β1, MMP-3 and MMP-9) and Th2 (IL-4, IL-13 and GATA3) relatedfactors were measured in the corneal epithelium and conjunctiva in thefollowing groups by real-time PCR.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5.0 software(GraphPad Inc). One-way analysis of variance (ANOVA) was used todetermine overall differences among groups, followed by a post-hoc test(Tukey’s post hoc). An unpaired t-test is used to evaluate statisticaldifferences between 2 experimental groups.

Example 47: HC-HA Activates IGF1-HIF1α-VEGF Signaling to PromoteAngiogenesis, Which is Further Promoted by Addtion of TGFβ1 in HumanCorneal Fibroblasts

In this example, the effect of HC-HA complexes on the induction ofangiogenic markers in human corneal fibroblasts was examined.

Human corneal fibroblasts (3000 cells/well in a 96-well plate) wereseeded on plastic dishes with or without immobilized HA, soluble HC-HA(PBS) (4X) or insoluble HC-HA (GnHCl) (4X) for 48 h as described above.The cells were then treated with or without TGFβ1 for 24 h before beingharvested for mRNA quantitation of IGF1, HIF1α and VEGF. Theexperimental groups were:

PBS PBS+TGF-β1 HA HA+TGF-β1 4X HC-HA PBS 4X HC-HA PBS+TGF-β1 4X HC-HA Gn4X HC-HA Gn+TGF-β1

Total RNAs were extracted using RNeasy Mini Kit (Qiagen) and werereverse transcribed using High Capacity Reverse Transcription Kit(Applied Biosystems). cDNA of each cell component was amplified byreal-time RT-PCR using specific primer-probe mixtures and DNA polymerasein 7000 Real-time PCR System (Applied Biosystems). Real-time RT-PCRprofile consisted of 10 minutes of initial activation at 95° C.,followed by 40 cycles of 15 seconds denaturation at 95° C., and 1 minuteannealing and extension at 60° C. The identity of each PCR product(IGF1, HIF1α and VEGF) was confirmed size determination using 2% agarosegels followed by ethidium bromide staining together with PCR markeraccording to EC3 Imaging System (BioImaging System).

HC-HA induced a 2- to 6-fold increase of IGF1 mRNA and 2-fold increaseof VEGF mRNA when the cells were in resting conditions (FIG. 92 ). Incontrast, HC-HA induced 5- to 12-fold increase of IGF1 mRNA and 5- to9-fold increase of VEGF mRNA when the cells were challenged by TGFβ (10ng/ml). VEGF has been demonstrated to be a major contributor toangiogenesis, increasing the number of capillaries in a given network.VEGF activation is controlled by upstream regulators such as IGF1 andHIF1α. Our results demonstrate that HC-HA activates IGF1-HIF1α-VEGFnetwork to promote angiogenesis, which is further promoted by addtion ofTGFβ1 in human corneal fibroblasts.

While preferred embodiments have been shown and described herein, itwill be obvious to those skilled in the art that such embodiments areprovided by way of example only. Numerous variations, changes, andsubstitutions may now occur. It should be understood that variousalternatives to the embodiments described herein can be employed inpracticing the described methods. It is intended that the followingclaims define the scope of the embodiments and that methods andstructures within the scope of these claims and their equivalents becovered thereby.

What is claimed: 1-148. (canceled)
 149. A method of treating a cancer inan individual in need thereof, the method comprising administering atherapeutically effective amount of a Heavy Chain-Hyaluronan/Pentraxin 3(HC-HA/PTX3) complex to the individual, thereby treating the cancer inthe individual.
 150. The method of claim 149, wherein the Hyaluronan(HA) of the HC-HA/PTX3 complex comprises high molecular weight (HMW) HA.151. The method of claim 149, wherein the HC-HA/PTX3 complex comprisesnative HC-HA/PTX3 complex (nHC-HA/PTX3).
 152. The method of claim 151,wherein the nHC-HA/PTX3 complex is isolated from fetal tissue that isfrozen or previously frozen.
 153. The method of claim 152, wherein thefetal tissue comprises amniotic membrane or umbilical cord extract. 154.The method of claim 152, wherein the nHC-HA/PTX3 complex is isolatedfrom amniotic membrane extract.
 155. The method of claim 149, whereinthe HC-HA/PTX3 complex comprises reconstituted HC-HA/PTX3(rcHC-HA/PTX3).
 156. The method of claim 155, wherein the rcHC-HA/PTX3comprises hyaluronan (HA), heavy chain 1 (HC1), and pentraxin 3 (PTX3).157. The method of claim 149, wherein the individual is a human. 158.The method of claim 149, wherein the treating comprises inhibiting thegrowth of cancer cells.
 159. The method of claim 149, wherein thetreating comprises killing cancer cells.
 160. The method of claim 149,wherein the cancer comprises prostate cancer, colorectal cancer, livercancer, kidney cancer, bladder cancer, brain cancer, ovarian cancer,lung cancer, melanoma, breast cancer, thyroid cancer, pancreatic cancer,stomach cancer, lung cancer, leukemia, lymphoma, sarcomas, gastriccancer, bone cancer, oral cancer, eye cancer, or carcinoma.
 161. Themethod of claim 149, wherein the cancer is pancreatic cancer.
 162. Themethod of claim 149, wherein the HC-HA/PTX3 complex is formulated foradministration by injection.
 163. The method of claim 149, wherein theHC-HA/PTX3 complex is formulated as a powder.
 164. The method of claim149, wherein the HC-HA/PTX3 complex is reconstituted with apharmaceutically acceptable excipient prior to injection.
 165. Themethod of claim 164, wherein the pharmaceutically acceptable excipientis selected from the group consisting of: a natural polymer, anadjuvant, a preservative, an agent for delaying absorption, a filler, abinder, an adsorbent, a buffer, and/or a combination thereof.